Dendritic cell receptor

ABSTRACT

The invention provides isolated human DEC-205, its extracellular domain and functionally equivalent fragments thereof. Also provided are polynucleotides encoding same and vectors which include such polynucleotides. Further provided are methods of recombinantly producing human DEC-205, an extracellular domain thereof or a functionally equivalent fragment, and ligands that bind to human DEC-205 or a fragment thereof. Also provided are constructs for use in prophylaxis or therapy comprising such a ligand, human DEC-205 or an extracellular domain thereof coupled to a toxin or to an antigen capable of inducing a protective immune response in a patient.

FIELD OF THE INVENTION

[0001] This invention relates to dendritic cell receptors. Inparticular, it relates to human DEC-205, to the production and usethereof, and to ligands which bind to it. Human DEC-205 and its ligandsare useful in prophylaxis and therapy.

BACKGROUND OF THE INVENTION

[0002] Dendritic cells perform important immunoregulatory functions bypresenting antigens in the form of peptides bound to cell-surface majorhistocompatibility complex (MHC) molecules to T cells. Identification ofthe mechanism by which this antigen presentation function is achievedtherefore has important implications in manipulating immune response inprophylaxis and therapy, particularly in humans.

[0003] Jiang et al, Nature 375: 151-155 (1995) disclose a murinedendritic cell receptor having a molecular weight of 205 kDa (murineDEC-205). However, they do not disclose a receptor on human dendriticcells.

[0004] The applicant has now identified a receptor on human dendriticcells. It is broadly to this receptor (likely to be the human homolog ofmurine DEC-205) that the present invention is directed.

SUMMARY OF THE INVENTION

[0005] The present invention has a number of aspects. In a first aspect,the invention provides isolated human DEC-205 which has an approximatemolecular weight of 198-205 kDa and which includes the following aminoacid sequences: (i) TVDCNDNQPGAICYYSGNETEKEVKPVDSVKCPSPVLNTPWIPFQNCCYNFIITKNRHMATTQDEVQSTCEKLHPKSHILSIRDEKENNFVLEQLLYFNYMASWVMLGITYRNNSL; and (ii)SQHRLFHLHSQKCLGLDITKSVNELRMFSCDSSAML;

[0006] or a functionally equivalent fragment thereof

[0007] In a further aspect, the invention provides isolated humanDEC-205 which comprises the amino acid sequence shown in FIG. 11 or afunctionally equivalent fragment thereof.

[0008] In a still further aspect, the invention provides isolated maturehuman DEC-205, which comprises the amino acids 27 to 1722 shown forhuman DEC-205 in FIG. 11.

[0009] In yet a further aspect, the invention provides an extracellulardomain of human DEC-205 or a functionally-equivalent fragment thereof.

[0010] In a preferred embodiment, the extracellular domain fragmentincludes at least a portion of carbohydrate recognition domain (CRD7),spacer, and a portion of carbohydrate recognition domain (CRD8) of humanDEC-205 (amino acids 1208 to 1323 of the amino acid sequence of FIG.11).

[0011] In a still further aspect, the invention provides apolynucleotide encoding human DEC-205 or its extracellular domain asdefined above. This polynucleotide is preferably DNA, more preferablycDNA, but can also be RNA.

[0012] In a specific embodiment, the polynucleotide coding for humanDEC-205 includes the following nucleotide sequences: (iii)   A ACA GTTGAT TGC AAT GAC AAT CAA CCA GGT GCT ATT TGC TAC TAT TCA GGA AAT GAG ACTGAA AAA GAG GTC AAA CCA GTT GAC AGT GTT AAA TGT CCA TCT CCT GTT CTA AATACT CCG TGG ATA CCA TTT CAG AAC TGT TGC TAC AAT TTC ATA ATA ACA AAG AATAGG CAT ATG GCA ACA ACA CAG GAT GAA GTT CAT ACT AAA TGC CAG AAA CTG AATCCA AAA TCA CAT ATT CTG AGT ATT CGA GAT GAA AAG GAG AAT AAC TTT GTT CTTGAG CAA CTG CTG TAC TTC AAT TAT ATG GCT TCA TGG GTC ATG TTA GGA ATA ACTTAT AGA AAT AAX TCT CTT; and (iv) ATT AAT ATG CTG TGG AAG TGG GTG TCCCAG CAT CGG CTC TTT CAT TTG CAC TCC CAA AAG TGC CTT GGC CTC GAT ATT ACCAAA TCG GTA AAT GAG CTG AGA ATG TTC AGC TGT GAC TCC AGT GCC ATG CTG TGGTGG AAA TGC GAG CAC CA

[0013] wherein X is T or G.

[0014] In a further embodiment, the polynucleotide comprises part or allof the nucleotide sequence of FIG. 10.

[0015] In yet a further aspect, the invention provides a vectorincluding a polynucleotide as defined above.

[0016] In still a further aspect, the invention provides a method ofproducing human DEC-205, the extracellular domain thereof or afunctional fragment comprising the steps of:

[0017] (a) culturing a host cell which has been transformed ortransfected with a vector as defined above to express the encoded humanDEC-205, extracellular domain or fragment; and

[0018] (b) recovering the expressed human DEC-205, extracellular domainor fragment.

[0019] As yet an additional aspect, the invention provides a ligand thatbinds to human DEC-205 or its extracellular domain as defined above.

[0020] Preferably, the ligand is an antibody or antibody bindingfragment or carbohydrate bearing protein.

[0021] The antibody or antibody binding fragment can be used in methodsfor extracting or isolating activated dendritic cells.

[0022] In still a further aspect, the invention provides a construct foruse in therapy or prophylaxis. The construct will usually be aligand-antigen construct or a DEC-205-antigen construct althoughligand-toxin and DEC-205-toxin constructs are also contemplated. Theligand-antigen construct preferably consists of an antibody or antibodybinding fragment which binds to human DEC-205 and a host-protectiveantigen. The DEC-205-antigen construct preferably consists of at leastthe extra-cellular domain of human DEC-205 and a host-protectiveantigen.

[0023] In yet further aspects, the invention contemplates methods oftherapy or prophylaxis which employ human DEC-205, ligands or constructscontaining them.

[0024] In yet a further aspect, the invention provides a molecule(hapten) which may be used to generate antibodies for identifying orpurifying human dendritic cells, which includes a peptide based uponpart or all of the sequence of FIG. 11.

DESCRIPTION OF THE DRAWINGS

[0025] While the invention is broadly as defined above, it will beappreciated by those persons skilled in this art that it is not limitedthereto and that it includes embodiments more particularly describedbelow. It will also be better understood by reference to theaccompanying drawings, in which

[0026]FIG. 1 shows the structure of human DEC-205;

[0027]FIG. 2 shows the strategy for isolation of human DEC-205 cDNA. A.A schematic presentation of human DEC-205 mRNA with the regionscorresponding to DEC-205 domains. The positions of the primers used forthe cDNA cloning and analysis are indicated with arrows. The positionsof reverse transcriptase-polymerase chain reaction (RT-PCR) fragments 1to 6 and the clone pBK14-1 are indicated with bars: B. RT-PCRamplification of fragment 1 and 2 from L428 and HEL cell line RNA. L428and HEL cells were subjected to RT-PCR with two pairs of degenerateprimers (DEC-a/-b, and DEC-d/-e), fractionated by electrophoresisthrough 2% agarose gel, and stained with ethidium bromide. C. RT-PCR and3′-RACE amplification of fragment 3 and 4 from L428 cells using theprimers 028/023 and 029/019, respectively. A cDNA pool of L428 cells wassubjected to 3′-RACE and RT-PCR, electrophoresed through 0.8% agarosegel, and stained with ethidium bromide. The numbers on the topcorrespond to the name of fragment in FIG. 2A. The positions of DNAmolecular size standard are indicateds to the right. The estimatedmolecular size of the RT-PCR products are indicated to the left;

[0028]FIG. 3 shows protein similarity between human and mouse DEC-205.A. The predicted amino acid sequence of human DEC-205 is aligned withthe mouse homolog. The regions corresponding to DEC-205 domain structureare bracketed. The positions of amino acids are shaded where there areidentical or conservatively replaced amino acids between the sequences,and the asterisks indicates conserved cysteines. The diamonds indicatespotential N-glycosylation sites conserved between the sequences. Thearrow indicates one amino acid deletion in CRD-5 of human DEC-205. Thecircles indicate conserved potential serine-phosphorylation sites byprotein kinase C (open circle) or casein kinase (closed circle). B. The% identity between human and mouse DEC-205 is indicated above eachdomain (boxed, See FIG. 2A for key);

[0029]FIG. 4 shows that human DEC-205 is probably a one-copy gene.Genomic DNA isolated from the peripheral blood of four individuals wasdigested with the restriction enzymes BgIII, BamHI, HindIII or EcoRI andsubjected to Southern blot analysis with the [³²P]cysteine-rich domainprobe. The final wash was 0.3×SSC at 65° C. The positions of the DNAmolecular size standards are indicated to the right;

[0030]FIG. 5 shows that human DEC-205 gene localizes on chromosome 2. Asomatic cell hybrid panel blot (restriction-digested with PstI) wassubjected to Southern blot analysis with the [³²P]cysteine-rich domainprobe. The final wash was 0.3×SSC at 65° C. The positions of the DNAmolecular size standards are indicated to the right. The estimatedmolecular size of the probe-specific bands are indicated to the left.The asterisk indicates weakly hybridized bands. M, male; F, female;

[0031]FIG. 6 shows that human DEC-205 gene maps to chromosome band 2q24.A. A metaphase spread of human chromosomes were subjected to fluorescentin situ hybridization (FISH) with 6.6 kb human DEC-205 cDNA probe. Thefinal wash was 0.1×SSC at 60° C. The FISH image was overlaid with aDAPI-stained chromosome image. The DEC-205 specific signals areindicated by the arrowheads. B. An inverted image of chromosome 2containing DEC-205-specific signal (see FIG. 6A) is aligned with anideogram of chromosome 2. The chromosome band corresponding to DEC-205gene is indicated to the right;

[0032]FIG. 7 shows that expression of DEC-205 transcripts within humanhematopoetic cell lines. Total RNA prepared from the cell lines weresubjected to Northern blot analysis with the [³²P]fragment 3 (A and B),or [³²P]-actin (C) probes. The final wash was 0.1×SSC at 65° C. Thepositions of the RNA molecular size standards are indicated to the rightThe estimated molecular size of DEC-205 transcripts are indicated to theleft. A, 24 h exposure; B, 72 h exposure;

[0033]FIG. 8 shows RT-PCR analysis of DEC-205 mRNA in human DCpreparations. Specific product is seen using lineage negative; fresh DC(lane 2) and a stronger signal with CMRF44⁺ low density cultured DC(lane 3). CD8⁺ T lymphocytes (lane 1) contain no DEC-205 mRNA Ethidiumstain.

[0034]FIG. 9 represents the result of an ELISA assay showing amonoclonal antibody binding specifically to DEC-205 peptide 1 and notpeptide 3. Positive control binding of a hyperimmunized rabbitanti-DEC-205-peptide 1 serum and hyperimmunized rabbitanti-DEC-205-peptide 2 serum are shown;

[0035]FIG. 10 gives the DNA sequence for human DEC-205 (coding regiononly);

[0036]FIG. 11 gives the human DEC-205 amino acid sequence.

DETAILED DESCRIPTION OF THE INVENTION

[0037] A. Human DEC-205

[0038] The human DEC-205 of the invention is believed to be the humanhomolog of murine DEC-205 and has an approximate molecular weight of 198to 205 kDa. It has the structure shown in FIGS. 1 and 2A. It also hasthe deduced amino acid sequence shown in FIG. 11.

[0039] Human DEC-205 can usefully be provided in a number of differentforms. These include human DEC-205 itself the “mature” form of humanDEC-205, and the extracellular receptor domain of human DEC-205.

[0040] The “mature” form of human DEC-205 of the invention is humanDEC-205 less its native amino-terminus leader or signal sequence,whereas the extracellular receptor domain is human DEC-205 lacking thesignal sequence, the transmembrane region and cytoplasmic domain (wherepresent).

[0041] The extracellular domain may be identified through commonlyrecognised criteria of extracellular amino acid sequences. Thedetermination of appropriate criteria is known to those skilled in theart, and has been described, for example by Hopp et al., Proc. Natl.Acad. Sci. U.S.A. 78, 3824-3828 (1991); Kyte et al., J. Mol. Biol. 157,105-132 (1982); Emini, J. Virol 55 836-839 (1985); Jameson et al. CABIOS 4 181-186 (1988); and Karplus et al. Naturwissenschaften 72,212-213 (1985). Amino acid domains predicted by these criteria to besurface exposed are characteristic of extracellular domains.

[0042] The amino acid sequences of the predicted regions for humanDEC-205 are shown in FIG. 3A. These include the amino acid sequences forthe signal peptide, cysteine-rich domain, fibronectin type II domain,Carbohydrate Recognition Domain-1, (CRD-1), CRD-2, CRD-3, CRD-4, CRD-5.CRD-6, CRD-7, CRD-8, CRD-9, CRD-10, transmembrane domain and cytoplasmicdomain.

[0043] Human DEC-205 of the invention or its extracellular receptordomain (or parts thereof) may be prepared by methods known in the art.Such methods include protein synthesis from individual amino acids asdescribed by Stuart and Young in “Solid-Phase Peptide Synthesis”, SecondEdition, Pierce Chemical Company (1984). It is however preferred thathuman DEC-205 and/or its extracellular receptor domain or parts thereofbe prepared by recombinant methods as will be detailed hereinafter.

[0044] Example 1 provides further details of human DEC-205.

EXAMPLE 1

[0045] Langerhans cells were prepared from human skin. Epidermal cellsuspensions were prepared from split thickness normal human breast skinby 30 min dispase (Boehringer-Mannheim, Mannheim, Germany; 0.5% in PBS)treatment at 37° C., followed by 10 min disaggregation in the presenceof trypsin (0.25% in PBS), DNase I (5U/ml in PBS) and 5 mM EDTA at roomtemperature. Langerhans cells were then enriched by Ficoll/Metrizoategradient separation (d=1.077 g/cm³). Final cell suspensions contained3-15% Langerhans cells as determined by HLA-DR positivity. Total RNA wasextracted using Trizol reagent according to the manufacturer'sinstructions.

[0046] Degenerate primers were prepared on an Applied Biosystems DNASynthesizer with the primer sequences (d) and (e) as set out below: (d)5′-GAX ACY GAX GGY TTX TGG AA-3′ (e) 3′-GCY GTX TTZ TCZ AAC CAC AT-5′

[0047] wherein X is C or T, Y is A, C, G or T, and Z is G or A.

[0048] Single stranded cDNA was prepared using total RNA and reversetranscribed by AMV reverse transcriptase using the 3′ primer (e).Subsequently, the cDNA was amplified using the 5′(d) and 3′(e) primerusing PCR amplification according to techniques known in the art.

[0049] The amplified products were run on a 2% agarose gel andvisualized with ethidium bromide staining.

[0050] The DNA was purified and ligated into the T tailed pGEM vector(available from Promega) using standard techniques. The ligation mixturewas transformed into competent E. coli JM 109 bacteria (available fromPromega) which were grown on agar plates with appropriate antibioticselection. Two colonies were isolated. DNA was prepared and digestedwith restriction enzymes. Two inserts of the same size as the PCRproduct were sequenced by double-stranded DNA sequencing techniquesusing a Sequence Kit (Sequence 2.0 USB Lab Supply, Pierce). The twoclones corresponded to human DEC-205.

[0051] The amino acid sequence of human DEC-205 was determined toinclude the following amino acid sequences: (i)TVDCNDNQPGAICYYSGNETEKEVKPVDSVKCPSPVLNTPWIPFQNCCYNFIITKNRHMATTQDEVQSTCEKLHPKSHILSIRDEKENNFVLEQLLYFNYMASWVMLGITYRNNSL; and (ii)SQHRLFHLHSQKCLGLDITKSVNELRMFSCDSSAML.

[0052] Determination of these sequences was fundamental to isolating thecDNA for human DEC-205 detailed below.

[0053] In the partial sequences given above, individual amino acids arerepresented by the single letter code as follows: Three-letterOne-letter Amino Acid abbreviation symbol Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic acid Asp D Asparagine or aspartic acid Asx BCysteine Cys C Glutamine Gln Q Glutamic Acid Glu E Glutamine or glutamicacid Glx Z Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu LLysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P SerineSer S Threonine Thr T Typtophan Trp W Tyrosine Tyr Y Valine Val VUnidentified X

[0054] This code also applies to the predicted full sequence of FIG. 11,deduced from the cDNA encoding human DEC-205 isolated as describedbelow.

[0055] B. Polynucleotides Encoding Human DEC-205

[0056] In another aspect of this invention, the applicants providepolynucleotides encoding human DEC-205 or its extracellular domain.These polynucleotides may be DNA (isolated from nature, synthesised orcDNA) or RNA. Most often, the polynucleotides will be DNA.

[0057] The polynucleotides of the invention specifically include thosewhich include the nucleotides (iii)   A ACA GTT GAT TGC AAT GAC AAT CAACCA GGT GCT ATT TGC TAC TAT TCA GGA AAT GAG ACT GAA AAA GAG GTC AAA CCAGTT GAC AGT GTT AAA TGT CCA TCT CCT GTT CTA AAT ACT CCG TGG ATA CCA TTTCAG AAC TGT TGC TAC AAT TTC ATA ATA ACA AAG AAT AGG CAT ATG GCA ACA ACACAG GAT GAA GTT CAT ACT AAA TGC CAG AAA CTG AAT CCA AAA TCA CAT ATT CTGAGT ATT CGA GAT GAA AAG GAG AAT AAC TTT GTT CTT GAG CAA CTG CTG TAC TTCAAT TAT ATG GCT TCA TGG GTC ATG TTA GGA ATA ACT TAT AGA AAT AAX TCT CTT;and (iv) ATT AAT ATG CTG TGG AAG TGG GTG TCC CAG CAT CGG CTC TTT CAT TTGCAC TCC CAA AAG TGC CTT GGC CTC GAT ATT ACC AAA TCG GTA AAT GAG CTG AGAATG TTC AGC TGT GAC TCC AGT GCC ATG CTG TGG TGG AAA TGC GAG CAC CA

[0058] wherein X is T or G, as well as the full nucleotide sequenceshown in FIG. 10, but are not limited thereto.

[0059] The invention also includes within its scope functionalequivalents of these polynucleotides.

[0060] This aspect of the invention will now be illustrated by thefollowing Examples.

EXAMPLE 2 Experimental Procedures

[0061] Cell culture—The cell lines, HEL, K562, KG-1, THP- 1, U937, Mannand Jurkat were obtained from the American Type Culture Collection(Rockville, Md.). L428 cells were provided by V. Diehl (Klinik forInnere Medizin, Cologne, Germany). HDLM2 and KMH2 cells were obtainedfrom the German Collection of Micro-organisms and Cell Culture(Braunscfweig, Germany). Mono Mac 6 cells (Bufler et al (1995) Eur. J.Immunol. 25, 604-610) were provided by H. Engelmann (Institute forImmunology, Munchen, Germany). All cell lines were maintained in RPMIi640, 10% fetal calf serum, 100 U/ml penicillin, 100 ug/ml streptomycinexcept that HDLM2 cells were with 20% fetal calf serum.

[0062] Isolation of leukocytes—Leukocyte populations were isolated usingstandard laboratory procedures.

[0063] Isolation of cDNA encoding for human DEC-205—A set of degenerateoligonucleotide primers were designed based on the published amino acidsequence of mouse DEC-205 (Jiang et al (1995), above) and synthesized inhouse or by Life Technologies (Auckland, New Zealand) (see FIG. 2A).These primers were:

[0064] DEC-a (5′-AAYATGCTNTGGAARTGGGT-3′),

[0065] DEC-b (5′-TGRTGYTCRCAYTTCCACCA-3′),

[0066] DEC-d (5′-GAYACNGAYGGNTTYTGGAA-3′) and

[0067] DEC-e (5′-GCNGTYTTRTCRAACCACAT-3′),

[0068] where Y=C or T, R=A or G, N=A or C or G or T. Total RNA isolatedfrom L428 or HEL cells was reverse transcribed with avian myeloblastosisvirus reverse transcriptase (Promega, Madison, Wis.) at 55° C. for 1 husing the primers DEC-b or DEC-e. PCR was performed using the resultantcDNA and Taq polymerase (Boehringer Mannheim, Auckland, New Zealand)with the primers DEC-a/-b for DEC-b-primed or DEC-d/-e for DEC-e-primedcDNAs. The PCR conditions used were the initial denaturation at 94° C.for 5 min, 35 cycles of denaturation at 94° C. for 1 min, annealing at54° C. for 1 min, extension at 72° C. for 1 min, and the final extensionat 72° C. for 5 min. The PCR reactions were fractionated with 2% agarosegel in 40 mM Tris-acetate, pH 8.3, 1 mM EDTA (TAE) buffer, and stainedwith 0.5 ug/ml ethidium bromide. The PCR fragments (fragment 1 and 2,see FIGS. 2A and 2B) were cloned into pGEM-T vector (Promega), andsequenced manually using Sequenase DNA sequencing kit (Amersham LifeScience, Auckland, New Zealand).

[0069] A set of oligonucleotide primers nested within the DNA sequenceof fragment 1 and 2 were synthesized (see FIG. 2A). These primers were:

[0070] 023 (5′-GCTCTAGAAACATGACCCATGAAGCC-3′ containing a XbaI site),

[0071] 028 (5′-GCTCTAGACATCGGCTCTTTCATITGT-3′ containing a XbaI site)and

[0072] 029 (5′-CGGGATTCACAGTTGATTGCAATGACA-3′ containing a EcoRI site)

[0073] where incorporated restriction sites are underlined. Two ug ofpoly(A) RNA from L428 cells was reverse transcribed with 200 U ofSuperScriptII (LifeTechnoloies) at 45° C. for 1 h using an oligo d(T)adaptor primer 018 (5′-GACTAGTCTGCAGAATTCTTTTTTTTTTTTTTTTT-3′,containing a SpeI PstI and EcoRI sites). After heat-inactivation at 70°C. for 15 min, the reaction was incubated with 1 U RNaseH (LifeTechnologies) at 37° C. for 30 min, heat-inactivated at 70° C. for 15min, and diluted to 1 ml with 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (L428cDNA pool). In order to isolate the fragment 3 (connecting the fragment1 and 2) (see FIG. 2A), PCR was performed with 5 ul of L428 cDNA pool,the primers 028 and 023, and 2.5 U of Expand enzyme mix (BoehringerMannheim). The PCR conditions were the initial denaturation at 94° C.for 2 min, 10 cycles of 10 cycles of denaturation at 94° C. for 15 sec,annealing at 53° C. for 30 sec, and extension at 68° C. for 4 min,followed by 20 cycles of denaturation at 94° C. for 15 sec, annealing at53° C. for 30 sec, and extension at 68° C. for 4 min plus additional 20sec for each cycle, and the final extension at 68° C. for 15 min.3′-rapid amplification of cDNA ends (3′-RACE) (Frohman et al (1988)Proc. Natl. Acad. Sci. U.S.A. 85, 8998-9002) was performed in order toisolate the fragment 4 (connecting the fragment 1 and the3′-untranslated region of DEC-205) (see FIG. 2A). PCR was performed with5 ul of L428 cDNA pool and the primer 029 and an adaptor primer 019(5′-GACTAGTCTGCAGAATTC, containing a SpeI, PstI and EcoRI site), in thesame conditions for the fragment 3. The PCR reactions were fractionatedwith 0.8% agarose gel in TAE buffer, and stained with ethidium bromide.Both the fragment 3 and 4 were restriction digested with XbaI and EcoRI,respectively, and cloned into pBluescript II (Stratagene, La Jolla,Calif.). The representative clones from the fragment 3 (pB38f1) and 4(pb30-3) were sequenced with a LI-COR automated sequencer (LI-COR,Lincoln, Nebr.) using SequiTherm cycle sequecing kit (EpicentreTechnologies, Madison, Wis.). If required, these plasmids were subjectedto exonucleaseIII-nested deletion using Erase-A-Base system (Promega),and used for sequencing.

[0074] An oligo dT-primed L428 cDNA library was prepared using ZAPExpress cDNA Gigapack Cloning kit (Stratagene) according tomanufacturer's instruction. The fragment 3 was labeled with [α-32P]dCTP(NEN) using Multiprime system (Amersham Life Science). The library wasscreened by plaque hybridization with the [³²P]fragment 3 using standardtechniques (Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual, 2Ed., Cold Spring HarbourLaboratory, New York, U.S.A.). The specific activity of the probe was0.8×10⁹ cpm/ug DNA and used at 1×10⁶ cpm/ml. The final wash was in0.1×SSC, 0.5% SDS at 65° C. (1×SSC is 0.15 M NaCl, 15 mMM Na-citrate,pH7.0). Positive clones were converted to phagemid pBK-CMV (Stratagene)and sequenced using an automated sequencer.

[0075] In order to verify the DNA sequence obtained from the PCR clones,pB38f for fragment 3 and pB30-3 for fragment 4, the fragment 5 wasPCR-amplified from a L428 cDNA pool using primers 058(5′-CGGGATCCCTCTGGCCGCGCACTAATGA-3′ containing a BamHI site) and 050(5′-CCGCTCGAGCTGTGGATACCAGCACATGCCT-3′ containing a XhoI site) (see FIG.2A). The PCR conditions were identical to that for the fragment 3 exceptusing longer extension period (6 min) for cycling. The fragment 5 wassequenced directly using the IRD₄₀-labeled custom primers (MWG-Biotech,Ebersberg, Germany) and a LI-COR automated sequencer without cloning.These primers were:

[0076] IRD001 (5′-GATGGGAACTCTTATGGGAGACCT-3′ at nucleotide 523-555),

[0077] IRD002 (5′-TGATGCAGGCTGGCTGCCAAATAA-3′ at nucleotide 1134-1157),

[0078] IRD003 (5′-AACTGGGCAACTGTTGGTGGAAGA-3′ at nucleotide 1759-1782),

[0079] IRD004 (5′-ATGGCGAAGAGGCTGGCATTTCTA-3′ at nucleotide 2334-2357),

[0080] IRD005 (5′-CTCAAGCAAGCGATACCTGTCACT-3′ at nucleotide 2972-2995),

[0081] IRD006 (5′-TGGGCAACTCGAAGACTGTGTAGT-3′ at nucleotide 3624-3647),

[0082] IRD007 (5′-CACCAGCACAGCATTCTTGCTTGT-3′ at nucleotide 4168-4191)and

[0083] IRD008 (5′-ATTTGTGAGCAGACTGATGAGGGA-3′ at nucleotide 4797-4820).

[0084] The sequences of these primers were based on those of pb38f1 andpb30-3, and they were positioned as 540-650 bp apart, ensuring thegeneration of contigs overlapping by at least 100 bp after automatedsequencing.

[0085] Southern blot analysis—Genomic DNA was prepared from peripheralblood of patients with hematological disorders (each patient waskaryotyped at Canterbury Health Laboratories, Christchurch, NewZealand). Approximately 8 ug of genomic DNA was digested with BgIII,BamHI, EcoRI, or HindIII, fractionated in 0.8% agarose gel in 89 mMTris-borate, pH 8.3, 2 mM EDTA, and transfered to Hybond N+ by capillaryreaction. A PCR-fragment corresponding to the cyteine-rich domain wasPCR-amplified from pBK14-1 using the primers 058 and 059(5′-CGGAATTCGATCTCATGATAAGGCTGGTCACA-3′ containing a EcoRI site) (seeFIG. 2A). Briefly, PCR was performed with 2 ng of pBK14-1, the primer058 and 059, and Taq polymerase. The PCR conditions used were theinitial denaturation at 94° C. for 2 min, 30 cycles of denaturation at94° C for 15 sec, annealing at 55° C. for 15 sec, extension at 72° C.for 30 sec, and the final extension at 72° C. for 5 min. The 450 bp PCRproduct was labeled with [α-³²P]dCTP using Multiprime labeling system(Amersham Life Science). The blot was hybridized with the probe usingstandard technique (Sambrook et al, (1989), above). The specificactivity of the probe was 0.8×10⁹ cpm/ug DNA and used at 1×10⁶ cpm/ml.The final wash was in 0.3×SSC, 0.5% SDS at 65° C., and exposed to X-OMATAR film (Kodak) with an intensifying screen at −70° C.

[0086] A blot containing PstI-digested genomic DNA from a human-rodentsomatic hybrid cell panel was obtained from Oncor (Gaithersburg, Md.),and probed with the [³²P]cysteine-rich domain fragment as describedabove.

[0087] Fluorescent in situ hybridization—Metaphase spreads were preparedfrom phytohaemagluttinin-stimulated peripheral blood lymphocytes of a46,XY male donor using standard cytogenetic procedures. The fragment 6was amplified by recombinant PCR with the fragment 3 and 4 (see FIG.2A). PCR was performed with each of the fragment 3 and 4 and the primers028 and 019 in the same conditions for the fragment 3 except usinglonger extension period (7 min) for cycling. The fragment 6 was labelledwith biotin-14-dCTP using a BioPrime random prime labelling kit(Bethesda Research Laboratories, Gathersberg, Md.), and hybridized tometaphase cells on slides. Conditions for hybridization andimmunofluorescent detection were essentially as described (Morris et al,(1993) Human Genetics, 91, 31-36), except that Cot 1 suppression was notrequired, slides were washed to a stringency of 0.1×SSC, 60° C. afterhybridization, and an additional amplification step was needed becauseof the small size of the probe. For precise chromosome bandlocalization, DAPI and FITC images were captured separately for eachmetaphase from the fluorescent microscope to computer using aPhotometrics KAF1400 CCD camera and IPLAB Spectrum software (SignalAnalytics, Va.), and colour-joined using Multiprobe extension software.

[0088] Northern blot analysis—Approximately 10 ug of total RNA fromcultured cells were fractionated in formaldehyde-denatured 1% agarosegel and transferred to Hybond N+ (Amersham) using 3 M NaCl, 8 mM NaOH, 2mM sarkosyl with Turboblotter (Schleicher & Schuell, Keene, N.H.) for 3h. The membrane was UV-crosslinked (Stratalinker, Stratagene), andhybridized with [³²P]fragment 3 or [³²P]human §-actin probe usingstandard techniques (Sambrook et al (1989), above). The specificactivity of the probes were 0.9-1.1×10⁹ cpm/ug DNA and used at0.7-1.1×10⁶ cpm/ml. The final wash was in 0.1×SSC, 0.5% SDS at 68° C.,and exposed to X-OMAT AR film (Kodak) with intensifying screen at −70°C.

[0089] Reverse transcription-PCR analysis—Total RNA from isolatedleukocytes was incubated with RNase-free DNasel (Life Technologies), andwas reverse transcribed using SuperscriptII with the oligo dT adaptorprimer 018. PCR was performed using a pair of DEC-205 specific primers060 (GTGGATCCAGTACAAGGGTCA at nucleotide 4655-4686) and 056(ACCAAATCAGTCCGCCCATGA at nucleotide 5116-5096) with Taq polymerase inthe presence of a PCR additive, Q buffer (Qiagen) by touch down PCR(Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., and Mattick, J.S., (1991) Nucleic Acid Res. 19, 4008). PCR conditions used were theinitial denaturation at 92° C. for 2 min, 21 cycles of denaturation at92° C. for 15 sec, annealing at 60° C. minus 0.5° C./cycle for 15 sec,extension at 68° C. for 30 sec, 15 cycles of denaturation at 92° C.,annealing at 50° C., extention at 68° C. for 1 min and the finalextension at 68° C. for 5 min. Human glycelaldehyde-3-phosphatedehydrogenase (GAPDH) (Tokunaga, K., Nakamura, Y., Sakata, K., Fujimori,K., Ohkubo, M., Sawada, K., and Sakiyama, S. (1987) Cancer Res. 47,5616-5619) was used for normalization. The primers for GAPDH were 053(ATGGGGAAGGTGAAGGTCGGA-3′ at nucleotide 61-81), and 055(AGGGGCCATCCACAGTCTTCT-3′ at nucleotide 634-614). The PCR reactions werefractionated with 1.5 % agarose gel in TAE buffer, and stained with 0.5ug/ml ethidium bromide.

[0090] Sequence data analysis—The National Center of BiotechnologyInformation (NCBI) Center electronic mail server BLAST was used tosearch for homologous sequences. Sequence alignments and motif searchwere done using Bestfit and Motifs programs, respectively, of GCGcomputer package (Madison, Wis.).

RESULTS

[0091] Isolation of cDNA for human DEC-205. Based on the amino acidsequence of mouse DEC-205, a set of degenerate primers were synthesizedand used to perform RT-PCR using the Hodgkin's disease-derived L428 cellline and the myeloid HEL cell lines (FIG. 2). The two pair of primers(DEC-d/-e, and DEC-a/-b) gave rise to the specific RT-PCR products,fragment 1 (390 bp) and 2 (150 bp), respectively (FIG. 2A and 2B). Thesespecific fragments were cloned and sequenced (data not shown). Thededuced amino acid sequences of fragment 1 and 2 were ˜80% identical tothat of mouse DEC-205, indicating that these fragments were derived fromthe cDNA of human DEC-205.

[0092] Primers nested within these fragments were synthesized andfurther RT-PCR and 3′-RACE performed using a L428 cDNA pool reversetranscribed with an oligo dT adapter primer 018. A 3.8 kb RT-PCR product(fragment 3) was obtained using primer 028 and 023 (FIGS. 2A and 2C). A3.2 kb 3′-RACE product (fragment 4) was obtained using primer 029 and anadaptor primer 019 (FIGS. 2A and 2C). The fragment 3 was cloned andseveral identical clones were identified by restriction enzyme mapanalysis (data not shown), and one of which, pb38f1, was fullysequenced: The DNA sequence of the fragment 3 (pB38f1) extending fromthe middle of cysteine-rich domain to the middle of CRD-8 (FIG. 2A), was82% identical to the published mouse DEC-205 cDNA sequence. The fragment4 was cloned and two distinct clones identified by restriction enzymemap analysis. Both clones were partially sequenced and the 3′ end DNAsequence of one clone (eg. pb30-3) was found to contain a poly A tail,and with 72% identical to 3′-untranslated region of mouse DEC-205 (datanot shown). Therefore, the pb30-3 was sequenced to obtain the DNAsequence of the coding region of DEC-205 plus partial 3′-untranslatedregion. The resulting DNA sequence for the coding region was ˜80%identical to that of mouse DEC-205 spanning from the middle of CRD-8 tothe end of cytoplasmic domain (FIG. 2A). The DNA sequences obtained frompb38f1 and pb30-3 overlapped by 320 bp, covering 95% of human DEC-205coding region.

[0093] In order to complete the 5′ end of the DEC-205 cDNA sequences aL428 cDNA library was screened by plaque hybridization using ³²P-labeledfragment 3 as a probe. A clone (pBK14-1) was isolated, and the 1.5 kbinsert of this clone was sequenced (FIG. 2A). The sequence was ˜80%identical to the mouse sequence and corresponded to the signal peptide,cysteine-rich domain, fibronectin type II domain, CRD-1 and part of theCRD-2. The pBK14-1 contained 51 bp 5′-untranslated region, andoverlapped with fragment 3 by ˜1.2 kb.

[0094] To validate the DNA sequence obtained from the PCR clones, afurther RT-PCR fragment (fragment 5) amplified with primers 058 (nestedin the cysteine-rich domain) and 050 (located ˜130 bp downstream of thestop codon) was prepared (FIG. 2A). The fragment 5 PCR product wassequenced directly using IRD₄₁-labeled custom primers without cloning. Atotal of 10 point mutations, presumably generated because of the lowfidelity of thermostable polymerases were found and corrected in the PCRclone-derived DNA sequence. The complete cDNA sequence for human DEC-205is 5166 bp in size, and encodes for a predicted 198 kDa type Itransmembrane protein with 1722 amino acids before post translationalmodification.

[0095] The deduced amino acid sequence of human DEC-205 showed 77%overall identity with the homologous mouse protein (FIG. 3A). All thecysteines, and putative N-glycosylation sites in the extracellulardomain of mouse DEC-205, were conserved in the human sequence. In thecytoplasmic domain the putative serine phosphorylation sites by proteinkinase C or casein kinase, and a tyrosine, which appears to be importantfor coated pit-mediated internalization (Ezekowitz, R. A. B., Sastry,K., Bailly, P., and Warner, A. (1990) J. Exp. Med. 172, 1785-1794; andZvaritch, E., Lambeau, G., and Lazdunski, M. (1996) J. Biol. Chem. 271,250-257), were also conserved. There was one amino acid deletion withinthe CRD-5 in human DEC-205. All the extracelluar domains, including thecysteine-rich domain, fibronectin type II domain, and CRD1-10 were74-87% identical between human and mouse sequences (FIG. 3B), suggestingthe importance of these domains for the function of DEC-205. Incontrast, the two hydrophobic domains, including the signal peptide andtransmembrane domain, showed much lower identity (57% and 52%,respectively (FIG. 3B)) with the mouse protein, confirming theobservation that these hydrohobic domains are more variable, and rapidlyevolved structures (Von Heijne, G. (1990) J. Membrane Biol. 115,195-201). DEC-205 is a single copy gene with polymorphism—Peripheralblood-derived genomic DNA from 4 individuals was restrictionenzyme-digested with BgIII, BamHI, HindIII or EcoRI, and subjected toSouthern blot analysis. The cysteine-rich domain of the macrophagemannose receptor (Kim, S. J., Ruiz, N., Bezouska, K., and Drickamer, K.(1992) Genomics 14, 721-727; and Harris, N., Peters, L. L., Eicher, E.M., Rits, M., Raspberry, D., Eichbaum, Q. G., Super, M., and Ezekowitz,R. A. B. (1994) Biochem. Biophys. Res. Com. 198, 682-692) andphospholipase A2 receptor (Ancian, P., Lambeau, G., Mattei, M. G., andLazdunski, M. (1995) 270, 8963-8970) is encoded by one exon. Therefore,we amplified the cysteine-rich domain of human DEC-205 using primers 058and 059 as a potential single exon probe (450 bp), and used this toprobe the Southern blot in high stringency. A single band appeared inBgIII-, BamHI- or HindIII-digested genomic DNA from all individuals,indicating that DEC-205 is a single copy gene (FIG. 4). The EcoRIdigests, however, produced a single band in two individuals and doublebands in another, indicating that the DEC-205 gene is polymorphic.Further Southern blot analysis with larger panel of individuals showedidentical results (data not shown). Therefore, DEC-205 is a single copygene with at least one polymorphic site.

[0096] DEC-205 gene maps to chromosome band 2q24—In order to map thehuman DEC-205 gene, a somatic cell hybrid panel Southern blot(PstI-digested) was probed with the [³²P]cysteine-rich domain asdescribed above (FIG. 5). A 3.0 kb band in human genomic DNA was foundto hybridize strongly, and the identical band appeared in chromosome2-containing somatic human-mouse hybrid cells, indicating that DEC-205gene localizes on chromosome 2. The probe also hybridized weakly withhamster DNA, suggesting the presence of DEC-205 homolog in hamster aswell as in the mouse (which also hybridized strongly). The origin of theweakly hybridized bands with apparent polymorphism in the humanDNA-containing lanes is not known. The identical band appeared inchromosome 2, and may either be related to an alternative exon structurefor this region of DEC-205 or result from weak cross hybridization toanother gene on chromosome 2.

[0097] Fluorescent in situ hybridization then was used to map theDEC-205 gene in detail (FIGS. 6A and 6B). The 6.4 kb recombinant PCRfragment (fragment 6) (FIG. 2A) was prepared from fragment 3 and 4,labeled with biotinylated nucleotides, and used as a probe in a highstringency (FIG. 6A). Ninety-one (80%) of a combined total 114 metaphasecells analysed from three experiments showed fluorescent signals on one(27) or both (64) chromosomes 2 in the middle of the long arm,specifically in band q24 (FIG. 6B). High resolution banding analysisprovided a more precise location of signals (not shown). No additionalsite-specific signals were detected on any other chromosome.

[0098] DEC-205 exhibits multiple transcripts in cell lines—A panel ofhuman cell lines, including myeloid, B lymphoid, T lymphoid andHodgkin's desease-derived cell lines, were analyzed for the expressionof DEC-205 transcripts by Northern blot analysis with the [³²P]fragment3 as a probe (FIGS. 7A and 7B). Two DEC-205 transcripts, 7.8 and 9.5 kbin size, were detected, and the 7.8 kb transcript was the most abundant.The expression level varied between cell lines, however the myeloid cellline THP-1, the B lymphoid cell line Mann and the Hodgkin's desease cellline KMH2 showed the highest level of expression. Even with longerexposure, DEC-205 transcripts were not detectable in K562, KG- 1,Monomac and Jurkat cells, suggesting these cells are DEC-205 negative(FIG. 7B). Interestingly all Hodgkin's disease-derived cell lines testedexpress the transcripts. Semiquantitative RT-PCR studies also supportthese results (data not shown).

[0099] C. Recombinant Expression of Human DEC-205

[0100] In yet another aspect, the present invention relates to therecombinant expression of human DEC-205 or of its extracellular domain.

[0101] The Polynucleotides that encode human DEC-205 or theextracellular domain of the invention may be inserted into known vectorsfor use in standard recombinant DNA techniques. Standard recombinant DNAtechniques are those such as are described in Sambrook et al.;“Molecular Cloning” 2nd Edition Cold Spring Harbour Laboratory Press(1987) and by Ausubel et al., Eds, “Current Protocols in MolecularBiology” Greene Publishing Associates and Wiley-Interscience, New York(1987).

[0102] Vectors for expressing proteins in bacteria, especially E. coli,are known. Such vectors include the PATH vectors described by Dieckmannand Tzagoloff in J. Biol. Chem. 260, 1513-1520 (1985). These vectorscontain DNA sequences that encode anthranilate synthetase (TrpE)followed by a polylinker at the carboxy terminus. Other expressionvector systems are based on beta-galactosidase (pGEX); lambda P maltosebinding protein (pMAL); and gluthathione S-transferase (PGST)—see Gene67, 31 (1988) and Peptide Research 3, 167 (1990).

[0103] Vectors useful in yeast and insect cells are available and wellknown. A suitable example of a yeast vector is the 2 μ plasmid.

[0104] Suitable vectors for use in mammalian cells are also known. Suchvectors include well-known derivatives of SV-40, adenovirus,retrovirus-derived DNA sequences and vectors derived from combination ofplasmids and phage DNA.

[0105] Further eucaryotic expression vectors are known in the art (e.g.P. J. Southern and P. Berg, J. Mol. Appl. Genet. 1, 327-341 (1982); S.Subramani et al, Mol. Cell. Biol. 1, 854-864 (1981); R. J. Kaufmann andP. A. Sharp, “Amplification And Expression of Sequences Cotransfectedwith a Modular Dihydrofolate Reductase Complementary DNA Gene,” J. Mol.Biol. 159, 601-621 (1982); R. J. Kaufmann and P. A. Sharp, Mol. Cell.Biol. 159 601-664 (1982); S. I. Scahill et al, “Expression AndCharacterization Of The Product Of A Human Immune Interferon DNA Gene InChinese Hamster Ovary Cells,” Proc. Natl. Acad. Sci. USA 80 46544659(1983); G. Urlaub and L. A. Chasin, Proc. Natl. Acad. Sci. USA 77,4216-4220, (1980).

[0106] The expression vectors useful in the present invention contain atleast one expression control sequence that is operatively linked to theDNA sequence or fragment to be expressed. The control sequence isinserted in the vector in order to control and to regulate theexpression of the cloned DNA sequence. Examples of useful expressioncontrol sequences are the lac system, the trp system, the tac system,the trc system, major operator and promoter regions of phage lambda, thecontrol region of fd coat protein, the glycolytic promoters of yeast,e.g. the promoter for 3-phosphoglycerate kinase, the promoters of yeastacid phosphatase, e.g. Pho5, the promoters of the yeast alpha-matingfactors, and promoters derived from polyoma, adenovirus, retrovirus, andsimian virus, e.g. the early and late promoters or SV40, and othersequences known to control the expression of genes of prokaryotic andeucaryotic cells and their viruses or combinations thereof.

[0107] Vectors containing the receptor-encoding DNA and control signalsare inserted into a host cell for expression of the receptor. Someuseful expression host cells include well-known prokaryotic andeucaryotic cells. Some suitable prokaryotic hosts include, for example,E. coli, such as E. coli SG-936. E. coli HB 101, E. coli W3 110, E. coliX1776, E. coli X2282, E. coli DHT, and E. coli MR01, Pseudomonas,Bacillus, such as Bacillus subtilis and Streptomyces. Suitableeucaryotic cells include yeast and other fungi, insect, animal cells,such as COS cells and CHO cells, human cells and plant cells in tissueculture.

[0108] D. Ligands

[0109] The invention also includes ligands that bind to human DEC-205 ofthe invention.

[0110] The ligand will usually be an antibody or an antibody bindingfragment raised against human DEC-205 or its extracellular domain, oragainst fragments thereof.

[0111] Such antibodies may be polyclonal but are preferably monoclonal.Monoclonal antibodies may be produced by methods known in the art. Thesemethods include the immunological method described by Kohler andMilstein in Nature 256 495497 (1975) and Campbell in “MonoclonalAntibody Technology, the Production and Characterization of Rodent andHuman Hybridomas” in Burdon et al. Eds, Laboratory Techniques inBiochemistry and Molecular Biology, Volume 13, Elsevier SciencePublishers, Amsterdam (1985); as well as by the recombinant DNA methoddescribed by Huse et al. in Science 246, 1275-1281 (1989).

[0112] In yet another form, the ligand may also be a non-protein,probably carbohydrate containing, molecule that acts as a ligand when itbinds to, or otherwise comes into contact with, human DEC-205.

[0113] In addition, ligands may be of two functional types. The firstfunctional type of ligand is a molecule which binds to human DEC-205 andstimulates it in performing its normal function (a “stimulant ligand”).The second functional type of ligand is a molecule which binds to humanDEC-205 and inhibits or prevents it performing its normal function (an“antagonistic ligand”).

[0114] Both types of ligand will find application in either therapeuticor prophylactic treatments as described below.

[0115] Example 3 describes the production of anti-DEC-205 antibodies.

EXAMPLE 3

[0116] Production of Anti-DEC-205 Antibodies

[0117] A BALB/c mouse was immunized ip/sc with L428 cells and boosted SCwith two peptides derived from the DEC-205 cDNA sequence. DEC-205peptide 1 ATTQDEVHTKC (aa1267-aa1277) and DEC-205-peptide 2TEKEVKPVDSVKC (aa1227-aa1239) were synthesized by Chiron Mimotopes PtyLtd (Clayton, Victoria, Australia). After a third immunization with thetwo DEC-205 peptides sc/ip/IV the mouse was sacrificed and a spleen cellsuspension prepared. The spleen cells were fused with the NS-1 myelomacell line using standard techniques (Hock et al, Immunology 1994;83:573). A hybridoma was subsequently isolated, 2F5, which producedmonoclonal antibody binding to the DEC-205-peptide 1 but not theDEC-205-peptide 2 or a third control DEC-205-peptide 3 (KCLGLDITKSVNELR)(aa82-aa96). This is shown by FIG. 9.

[0118] E. Constructs

[0119] The invention also provides constructs. The constructs willgenerally include antigens against which an immune response is desiredbut can also include other products to be delivered specifically todendritic cells. Toxins, such as the ricin A chain are not excluded. Theother component of the construct will vary, being either a ligand asdescribed above or at least the extracellular domain of human DEC-205.Both constructs will have the potential to manipulate the immune systemof the host.

[0120] In the ligand-antigen constructs, ligands which bind to humanDEC-205 (usually antibodies, antibody-binding fragments or carbohydratesexpressing proteins) can be coupled or otherwise associated with theantigen against which an immune response is desired. An example of suchantigens are sugar-coated antigens such as tumour-associated antigens Inuse, the ligand component binds to human DEC-205 and the dendritic cellis ‘primed’ with the associated antigen. This ‘priming’ action willassist in the induction of an immediate immune response against theantigen.

[0121] The ligand-antigen construct can take any appropriate form foradministration to the dendritic cells. Such forms may differ dependingupon whether the therapeutic protocol involves isolation of the patientsdendritic cells (so that the priming action can take place in vitro) orwhether the construct is to be administered to a patient in vivo.

[0122] The construct can be directly administered to a patient for invivo treatment. It can also be administered in a form which allows theconstruct to be expressed within the patient.

[0123] One example of such a form for administration to a patient invivo is a live recombinant viral vaccine. Such a vaccine includes apolynucleotide encoding the DEC-205 ligand (or a portion thereof) andthe antigen. The vaccine is administered to the patient and, once withinthe patient, expresses the encoded ligand and antigen to bind to thepatients dendritic cells (via human DEC-205).

[0124] A number of such live recombinant viral vaccine systems areknown. An example of such a system is the Vaccinia virus system (U.S.Pat. No. 4,603,112; Brochier et al., Nature 354:520 (1991)).

[0125] Administration can be via intravenous, intramuscular,subcutaneous, topical, oral intra nasal, rectal orintracerebroventricular routes, as appropriate.

[0126] F. Applications

[0127] Human DEC-205, its ligands and the constructs discussed above canbe employed therapeutically or prophylactically in accordance with thisinvention to promote or inhibit any of the known actions of dendriticcells and/or to manipulate the immune system.

[0128] Thus, the antagonistic ligands per se have potential applicationinter alia blocking or inhibiting the immune response duringtransplantation procedures.

[0129] Ligands also have application in delivering other products withwhich they are associated directly to dendritic cells. This can be fortherapeutic purposes (where the delivered product is an immunogenicantigen) as discussed above. It can also be to target a toxin (such asthe ricin A-chain specifically to dendritic cells to selectively destroythem as part of an immunosuppressive process.

[0130] G. The Use of Human DEC-205 to Detect Dendritic Cells in CellSuspensions on Tissues and to Purify Dendritic Cells

[0131] Monoclonal antibodies or other ligands binding to DEC-205 may beused to identify or isolate DC for scientific study or therapeuticapplication. For this application, the antibodies or ligands can be usedin conjunction with conventional identification/separation systems. Anexample of such a system is the avidin-biotin immunoaffinity systemavailable from Cell-Pro Inc, Wash., U.S.A. (see U.S. Pat. No. 5,215,927,U.S. Pat. No. 5,225,353, U.S. Pat. No. 5,262,334 and U.S. Pat. No.5,240,856).

[0132] This system employs directly or indirectly a biotinylatedmonoclonal antibody directed against a target cell and a columncontaining immunobilized avidin and can be readily adapted to extractactivated human dendritic cells, in this case from human peripheralblood, using the anti-DEC-205 antibody as follows:

[0133] 1. A sample of human peripheral blood containing the humandendritic cells is mixed with biotinylated anti-DEC-205 antibody andincubated to allow formation of antibody/human DC complexes.

[0134] 2. Following incubation, the mixture is introduced into a CellProcontinuous-flow immunoadsorption column filled with avidin-coated beads,the strong affinity between biotin and avidin causing the biotin-coatedantibodies (together with the human DC to which they have bound) toadhere to the avidin-coated beads.

[0135] 3. After unwanted cells present in the mixture are washed away,captured activated human DC are removed from the column by gentleagitation and are available for use.

[0136] Variations on this theme using the anti-DEC-205 antibody asprimary antibody (to bind to activated DC) and a biotinylated secondaryantibody (to bind to the anti-DEC-205 antibody) can also be employed.

[0137] It will be appreciated that before admixture with theanti-DEC-205 antibody in accordance with the above protocol the humanperipheral blood sample should be treated to ensure that the DC thesample contains are activated. This can easily be achieved by, forexample, overnight incubation of the sample.

[0138] H. Functional Equivalents

[0139] The invention includes functional equivalents of human DEC-205,extracellular domains and nucleic acid molecules described above.

[0140] Human DEC-205 and its extracellular domain are or includeproteins. A protein is considered a functional equivalent of anotherprotein for a specific function if the equivalent protein isimmunologically cross-reactive with, and has the same function as, theoriginal protein. The equivalent may, for example, be a fragment of theprotein, or a substitution, addition or deletion mutant of the protein.

[0141] For example, it is possible to substitute amino acids in asequence with equivalent amino acids using conventional techniques.Groups of amino acids known normally to be equivalent are:

[0142] (a) Ala(A) Ser(S) Thr(T) Pro(P) Gly(G);

[0143] (b) Asn(N) Asp(D) Glu(E) Gln(Q);

[0144] (c) His(H) Arg(R) Lys(K);

[0145] (d) Met(M) Leu(L) Ile(I) Val(V); and

[0146] (e) Phe(F) Tyr(Y) Trp(W).

[0147] Substitutions, additions and/or deletions in human DEC-205 may bemade as long as the resulting equivalent protein is immunologicallycross-reactive with, and have the same function as, the native humanDEC-205.

[0148] The equivalent human DEC-205 will normally have substantially thesame amino acid sequence as the native human DEC-205. An amino acidsequence that is substantially the same as another sequence, but thatdiffers from the other sequence by means of one or more substitutions,additions and/or deletions is considered to be an equivalent sequence.Preferably, less than 25%, more preferably less than 10%, and mostpreferably less than 5% of the number of amino acid residues in theamino acid sequence of the native human DEC-205 are substituted for,added to, or deleted from

[0149] Equivalent nucleic acid molecules include nucleic acid sequencesthat encode proteins equivalent to human DEC-205 as defined above.Equivalent nucleic acid molecules also include nucleic acid sequencesthat, due to the degeneracy of the nucleic acid code, differ from nativenucleic acid sequences in ways that do not affect the correspondingamino acid sequences.

[0150] Those persons skilled in the art will of course appreciate thatthe above description is provided by way of example only and that theinvention is limited only by the lawful scope of the appended claims.

1 31 1 1722 PRT Homo sapiens 1 Met Arg Thr Gly Trp Ala His Pro Ser ProPro Gly Gly Ala Pro His 1 5 10 15 Ala Ala Leu Leu Val Leu Arg Ser ArgGly Ala Leu Trp Pro Arg Thr 20 25 30 Asn Asp Pro Phe Thr Ile Val His GlyAsn Thr Gly Lys Cys Ile Lys 35 40 45 Pro Val Tyr Gly Trp Ile Val Ala AspAsp Cys Asp Glu Thr Glu Asp 50 55 60 Lys Leu Trp Lys Trp Val Ser Gln HisArg Leu Phe His Leu His Ser 65 70 75 80 Gln Lys Cys Leu Gly Leu Asp IleThr Lys Ser Val Asn Glu Leu Arg 85 90 95 Met Phe Ser Cys Asp Ser Ser AlaMet Leu Trp Trp Lys Cys Glu His 100 105 110 His Ser Leu Tyr Gly Ala AlaArg Tyr Trp Leu Ala Leu Lys Asp Gly 115 120 125 His Gly Thr Ala Ile SerAsn Ala Ser Asp Val Trp Lys Lys Gly Gly 130 135 140 Ser Glu Glu Ser LeuCys Asp Gln Pro Tyr His Glu Ile Tyr Thr Arg 145 150 155 160 Asp Gly AsnSer Tyr Gly Arg Pro Cys Glu Phe Pro Phe Leu Ile Asp 165 170 175 Gly ThrTrp His His Asp Cys Ile Leu Asp Glu Asp His Ser Gly Pro 180 185 190 TrpCys Ala Thr Thr Leu Asn Tyr Glu Tyr Asp Arg Lys Trp Gly Ile 195 200 205Cys Leu Lys Pro Glu Asn Gly Cys Glu Asp Asn Trp Glu Lys Asn Glu 210 215220 Gln Phe Gly Ser Cys Tyr Gln Phe Asn Thr Gln Thr Ala Leu Ser Trp 225230 235 240 Lys Glu Ala Tyr Val Ser Cys Gln Asn Gln Gly Ala Asp Leu LeuSer 245 250 255 Ile Asn Ser Ala Ala Glu Leu Thr Tyr Leu Lys Glu Lys GluGly Ile 260 265 270 Ala Lys Ile Phe Trp Ile Gly Leu Asn Gln Leu Tyr SerAla Arg Gly 275 280 285 Trp Glu Trp Ser Asp His Lys Pro Leu Asn Phe LeuAsn Trp Asp Pro 290 295 300 Asp Arg Pro Ser Ala Pro Thr Ile Gly Gly SerSer Cys Ala Arg Met 305 310 315 320 Asp Ala Glu Ser Gly Leu Trp Gln SerPhe Ser Cys Glu Ala Gln Leu 325 330 335 Pro Tyr Val Cys Arg Lys Pro LeuAsn Asn Thr Val Glu Leu Thr Asp 340 345 350 Val Trp Thr Tyr Ser Asp ThrArg Cys Asp Ala Gly Trp Leu Pro Asn 355 360 365 Asn Gly Phe Cys Tyr LeuLeu Val Asn Glu Ser Asn Ser Trp Asp Lys 370 375 380 Ala His Ala Lys CysLys Ala Phe Ser Ser Asp Leu Ile Ser Ile His 385 390 395 400 Ser Leu AlaAsp Val Glu Val Val Val Thr Lys Leu His Asn Glu Asp 405 410 415 Ile LysGlu Glu Val Trp Ile Gly Leu Lys Asn Ile Asn Ile Pro Thr 420 425 430 LeuPhe Gln Trp Ser Asp Gly Thr Glu Val Thr Leu Thr Tyr Trp Asp 435 440 445Glu Asn Glu Pro Asn Val Pro Tyr Asn Lys Thr Pro Asn Cys Val Ser 450 455460 Tyr Leu Gly Glu Leu Gly Gln Trp Lys Val Gln Ser Cys Glu Glu Lys 465470 475 480 Leu Lys Tyr Val Cys Lys Arg Lys Gly Glu Lys Leu Asn Asp AlaSer 485 490 495 Ser Asp Lys Met Cys Pro Pro Asp Glu Gly Trp Lys Arg HisGly Glu 500 505 510 Thr Cys Tyr Lys Ile Tyr Glu Asp Glu Val Pro Phe GlyThr Asn Cys 515 520 525 Asn Leu Thr Ile Thr Ser Arg Phe Glu Gln Glu TyrLeu Asn Asp Leu 530 535 540 Met Lys Lys Tyr Asp Lys Ser Leu Arg Lys TyrPhe Trp Thr Gly Leu 545 550 555 560 Arg Asp Val Asp Ser Cys Gly Glu TyrAsn Trp Ala Thr Val Gly Gly 565 570 575 Arg Arg Arg Ala Val Thr Phe SerAsn Trp Asn Phe Leu Glu Pro Ala 580 585 590 Ser Pro Gly Gly Cys Val AlaMet Ser Thr Gly Lys Ser Val Gly Lys 595 600 605 Trp Glu Val Lys Asp CysArg Ser Phe Lys Ala Leu Ser Ile Cys Lys 610 615 620 Lys Met Ser Gly ProLeu Gly Pro Glu Glu Ala Ser Pro Lys Pro Asp 625 630 635 640 Asp Pro CysPro Glu Gly Trp Gln Ser Phe Pro Ala Ser Leu Ser Cys 645 650 655 Tyr LysVal Phe His Ala Glu Arg Ile Val Arg Lys Arg Asn Trp Glu 660 665 670 GluAla Glu Arg Phe Cys Gln Ala Leu Gly Ala His Leu Ser Ser Phe 675 680 685Ser His Val Asp Glu Ile Lys Glu Phe Leu His Phe Leu Thr Asp Gln 690 695700 Phe Ser Gly Gln His Trp Leu Trp Ile Gly Leu Asn Lys Arg Ser Pro 705710 715 720 Asp Leu Gln Gly Ser Trp Gln Trp Ser Asp Arg Thr Pro Val SerThr 725 730 735 Ile Ile Met Pro Asn Glu Phe Gln Gln Asp Tyr Asp Ile ArgAsp Cys 740 745 750 Ala Ala Val Lys Val Phe His Arg Pro Trp Arg Arg GlyTrp His Phe 755 760 765 Tyr Asp Asp Arg Glu Phe Ile Tyr Leu Arg Pro PheAla Cys Asp Thr 770 775 780 Lys Leu Glu Trp Val Cys Gln Ile Pro Lys GlyArg Thr Pro Lys Thr 785 790 795 800 Pro Asp Trp Tyr Asn Pro Asp Arg AlaGly Ile His Gly Pro Pro Leu 805 810 815 Ile Ile Glu Gly Ser Glu Tyr TrpPhe Val Ala Asp Leu His Leu Asn 820 825 830 Tyr Glu Glu Ala Val Leu TyrCys Ala Ser Asn His Ser Phe Leu Ala 835 840 845 Thr Ile Thr Ser Phe ValGly Leu Lys Ala Ile Lys Asn Lys Ile Ala 850 855 860 Asn Ile Ser Gly AspGly Gln Lys Trp Trp Ile Arg Ile Ser Glu Trp 865 870 875 880 Pro Ile AspAsp His Phe Thr Tyr Ser Arg Tyr Pro Trp His Arg Phe 885 890 895 Pro ValThr Phe Gly Glu Glu Cys Leu Tyr Met Ser Ala Lys Thr Trp 900 905 910 LeuIle Asp Leu Gly Lys Pro Thr Asp Cys Ser Thr Lys Leu Pro Phe 915 920 925Ile Cys Glu Lys Tyr Asn Val Ser Ser Leu Glu Lys Tyr Ser Pro Asp 930 935940 Ser Ala Ala Lys Val Gln Cys Ser Glu Gln Trp Ile Pro Phe Gln Asn 945950 955 960 Lys Cys Phe Leu Lys Ile Lys Pro Val Ser Leu Thr Phe Ser GlnAla 965 970 975 Ser Asp Thr Cys His Ser Tyr Gly Gly Thr Leu Pro Ser ValLeu Ser 980 985 990 Gln Ile Glu Gln Asp Phe Ile Thr Ser Leu Leu Pro AspMet Glu Ala 995 1000 1005 Thr Leu Trp Ile Gly Leu Arg Trp Thr Ala TyrGlu Lys Ile Asn Lys 1010 1015 1020 Trp Thr Asp Asn Arg Glu Leu Thr TyrSer Asn Phe His Pro Leu Leu 1025 1030 1035 1040 Val Ser Gly Arg Leu ArgIle Pro Glu Asn Phe Phe Glu Glu Glu Ser 1045 1050 1055 Arg Tyr His CysAla Leu Ile Leu Asn Leu Gln Lys Ser Pro Phe Thr 1060 1065 1070 Gly ThrTrp Asn Phe Thr Ser Cys Ser Glu Arg His Phe Val Ser Leu 1075 1080 1085Cys Gln Lys Tyr Ser Glu Val Lys Ser Arg Gln Thr Leu Gln Asn Ala 10901095 1100 Ser Glu Thr Val Lys Tyr Leu Asn Asn Leu Tyr Lys Ile Ile ProLys 1105 1110 1115 1120 Thr Leu Thr Trp His Ser Ala Lys Arg Glu Cys LeuLys Ser Asn Met 1125 1130 1135 Gln Leu Val Ser Ile Thr Asp Pro Tyr GlnGln Ala Phe Leu Ser Val 1140 1145 1150 Gln Ala Leu Leu His Asn Ser SerLeu Trp Ile Gly Leu Phe Ser Gln 1155 1160 1165 Asp Asp Glu Leu Asn PheGly Trp Ser Asp Gly Lys Arg Leu His Phe 1170 1175 1180 Ser Arg Trp AlaGlu Thr Asn Gly Gln Leu Glu Asp Cys Val Val Leu 1185 1190 1195 1200 AspThr Asp Gly Phe Trp Lys Thr Val Asp Cys Asn Asp Asn Gln Pro 1205 12101215 Gly Ala Ile Cys Tyr Tyr Ser Gly Asn Glu Thr Glu Lys Glu Val Lys1220 1225 1230 Pro Val Asp Ser Val Lys Cys Pro Ser Pro Val Leu Asn ThrPro Trp 1235 1240 1245 Ile Pro Phe Gln Asn Cys Cys Tyr Asn Phe Ile IleThr Lys Asn Arg 1250 1255 1260 His Met Ala Thr Thr Gln Asp Glu Val HisThr Lys Cys Gln Lys Leu 1265 1270 1275 1280 Asn Pro Lys Ser His Ile LeuSer Ile Arg Asp Glu Lys Glu Asn Asn 1285 1290 1295 Phe Val Leu Glu GlnLeu Leu Tyr Phe Asn Tyr Met Ala Ser Trp Val 1300 1305 1310 Met Leu GlyIle Thr Tyr Arg Asn Asn Ser Leu Met Trp Phe Asp Lys 1315 1320 1325 ThrPro Leu Ser Tyr Thr His Trp Arg Ala Gly Arg Pro Thr Ile Lys 1330 13351340 Asn Glu Lys Phe Leu Ala Gly Leu Ser Thr Asp Gly Phe Trp Asp Ile1345 1350 1355 1360 Gln Thr Phe Lys Val Ile Glu Glu Ala Val Tyr Phe HisGln His Ser 1365 1370 1375 Ile Leu Ala Cys Lys Ile Glu Met Val Asp TyrLys Glu Glu His Asn 1380 1385 1390 Thr Thr Leu Pro Gln Phe Met Pro TyrGlu Asp Gly Ile Tyr Ser Val 1395 1400 1405 Ile Gln Lys Lys Val Thr TrpTyr Glu Ala Leu Asn Met Cys Ser Gln 1410 1415 1420 Ser Gly Gly His LeuAla Ser Val His Asn Gln Asn Gly Gln Leu Phe 1425 1430 1435 1440 Leu GluAsp Ile Val Lys Arg Asp Gly Phe Pro Leu Trp Val Gly Leu 1445 1450 1455Ser Ser His Asp Gly Ser Glu Ser Ser Phe Glu Trp Ser Asp Gly Ser 14601465 1470 Thr Phe Asp Tyr Ile Pro Trp Lys Gly Gln Thr Ser Pro Gly AsnCys 1475 1480 1485 Val Leu Leu Asp Pro Lys Gly Thr Trp Lys His Glu LysCys Asn Ser 1490 1495 1500 Val Lys Asp Gly Ala Ile Cys Tyr Lys Pro ThrLys Ser Lys Lys Leu 1505 1510 1515 1520 Ser Arg Leu Thr Tyr Ser Ser ArgCys Pro Ala Ala Lys Glu Asn Gly 1525 1530 1535 Ser Arg Trp Ile Gln TyrLys Gly His Cys Tyr Lys Ser Asp Gln Ala 1540 1545 1550 Leu His Ser PheSer Glu Ala Lys Lys Leu Cys Ser Lys His Asp His 1555 1560 1565 Ser AlaThr Ile Val Ser Ile Lys Asp Glu Asp Glu Asn Lys Phe Val 1570 1575 1580Ser Arg Leu Met Arg Glu Asn Asn Asn Ile Thr Met Arg Val Trp Leu 15851590 1595 1600 Gly Leu Ser Gln His Ser Val Asp Gln Ser Trp Ser Trp LeuAsp Gly 1605 1610 1615 Ser Glu Val Thr Phe Val Lys Trp Glu Asn Lys SerLys Ser Gly Val 1620 1625 1630 Gly Arg Cys Ser Met Leu Ile Ala Ser AsnGlu Thr Trp Lys Lys Val 1635 1640 1645 Glu Cys Glu His Gly Phe Gly ArgVal Val Cys Lys Val Pro Leu Gly 1650 1655 1660 Pro Asp Tyr Thr Ala IleAla Ile Ile Val Ala Thr Leu Ser Ile Leu 1665 1670 1675 1680 Val Leu MetGly Gly Leu Ile Trp Phe Leu Phe Gln Arg His Arg Leu 1685 1690 1695 HisLeu Ala Gly Phe Ser Ser Val Arg Tyr Ala Gln Gly Val Asn Glu 1700 17051710 Asp Glu Ile Met Leu Pro Ser Phe His Asp 1715 1720 2 5169 DNA Homosapiens CDS (1)..(5166) 2 atg agg aca ggc tgg gcg cac ccc tcg ccg cccggc ggg gct cct cat 48 Met Arg Thr Gly Trp Ala His Pro Ser Pro Pro GlyGly Ala Pro His 1 5 10 15 gct gct ctt ctg gtt ctt cga tct cgc gga gccctc tgg ccg cgc act 96 Ala Ala Leu Leu Val Leu Arg Ser Arg Gly Ala LeuTrp Pro Arg Thr 20 25 30 aat gac ccc ttc acc atc gtc cat gga aat acg ggcaag tgc atc aag 144 Asn Asp Pro Phe Thr Ile Val His Gly Asn Thr Gly LysCys Ile Lys 35 40 45 cca gtg tat ggc tgg ata gta gca gac gac tgt gat gaaact gag gac 192 Pro Val Tyr Gly Trp Ile Val Ala Asp Asp Cys Asp Glu ThrGlu Asp 50 55 60 aag tta tgg aag tgg gtg tcc cag cat cgg ctc ttt cat ttgcac tcc 240 Lys Leu Trp Lys Trp Val Ser Gln His Arg Leu Phe His Leu HisSer 65 70 75 80 caa aag tgc ctt ggc ctc gat att acc aaa tcg gta aat gagctg aga 288 Gln Lys Cys Leu Gly Leu Asp Ile Thr Lys Ser Val Asn Glu LeuArg 85 90 95 atg ttc agc tgt gac tcc agt gcc atg ctg tgg tgg aaa tgt gagcac 336 Met Phe Ser Cys Asp Ser Ser Ala Met Leu Trp Trp Lys Cys Glu His100 105 110 cac tct ctg tac gga gct gcc cgg tac tgg ctg gct ctg aag gatgga 384 His Ser Leu Tyr Gly Ala Ala Arg Tyr Trp Leu Ala Leu Lys Asp Gly115 120 125 cat ggc aca gca atc tca aat gca tct gat gtc tgg aag aaa ggaggc 432 His Gly Thr Ala Ile Ser Asn Ala Ser Asp Val Trp Lys Lys Gly Gly130 135 140 tca gag gaa agc ctt tgt gac cag cct tat cat gag atc tat accaga 480 Ser Glu Glu Ser Leu Cys Asp Gln Pro Tyr His Glu Ile Tyr Thr Arg145 150 155 160 gat ggg aac tct tat ggg aga cct tgt gaa ttt cca ttc ttaatt gat 528 Asp Gly Asn Ser Tyr Gly Arg Pro Cys Glu Phe Pro Phe Leu IleAsp 165 170 175 ggg acc tgg cat cat gat tgc att ctt gat gaa gat cat agtggg cca 576 Gly Thr Trp His His Asp Cys Ile Leu Asp Glu Asp His Ser GlyPro 180 185 190 tgg tgt gcc acc acc tta aat tat gaa tat gac cga aag tggggc atc 624 Trp Cys Ala Thr Thr Leu Asn Tyr Glu Tyr Asp Arg Lys Trp GlyIle 195 200 205 tgc tta aag cct gaa aac ggt tgt gaa gat aat tgg gaa aagaac gag 672 Cys Leu Lys Pro Glu Asn Gly Cys Glu Asp Asn Trp Glu Lys AsnGlu 210 215 220 cag ttt gga agt tgc tac caa ttt aat act cag acg gct ctttct tgg 720 Gln Phe Gly Ser Cys Tyr Gln Phe Asn Thr Gln Thr Ala Leu SerTrp 225 230 235 240 aaa gaa gct tat gtt tca tgt cag aat caa gga gct gattta ctg agc 768 Lys Glu Ala Tyr Val Ser Cys Gln Asn Gln Gly Ala Asp LeuLeu Ser 245 250 255 atc aac agt gct gct gaa tta act tac ctt aaa gaa aaagaa ggc att 816 Ile Asn Ser Ala Ala Glu Leu Thr Tyr Leu Lys Glu Lys GluGly Ile 260 265 270 gct aag att ttc tgg att ggt tta aat cag cta tac tctgct aga ggc 864 Ala Lys Ile Phe Trp Ile Gly Leu Asn Gln Leu Tyr Ser AlaArg Gly 275 280 285 tgg gaa tgg tca gac cac aaa cca tta aac ttt ctc aactgg gat cca 912 Trp Glu Trp Ser Asp His Lys Pro Leu Asn Phe Leu Asn TrpAsp Pro 290 295 300 gac agg ccc agt gca cct act ata ggt ggc tcc agc tgtgca aga atg 960 Asp Arg Pro Ser Ala Pro Thr Ile Gly Gly Ser Ser Cys AlaArg Met 305 310 315 320 gat gct gag tct ggt ctg tgg cag agc ttt tcc tgtgaa gct caa ctg 1008 Asp Ala Glu Ser Gly Leu Trp Gln Ser Phe Ser Cys GluAla Gln Leu 325 330 335 ccc tat gtc tgc agg aaa cca tta aat aat aca gtggag tta aca gat 1056 Pro Tyr Val Cys Arg Lys Pro Leu Asn Asn Thr Val GluLeu Thr Asp 340 345 350 gtc tgg aca tac tca gat acc cgc tgt gat gca ggctgg ctg cca aat 1104 Val Trp Thr Tyr Ser Asp Thr Arg Cys Asp Ala Gly TrpLeu Pro Asn 355 360 365 aat gga ttt tgc tat ctg ctg gta aat gaa agt aattcc tgg gat aag 1152 Asn Gly Phe Cys Tyr Leu Leu Val Asn Glu Ser Asn SerTrp Asp Lys 370 375 380 gca cat gcg aaa tgc aaa gcc ttc agt agt gac ctaatc agc att cat 1200 Ala His Ala Lys Cys Lys Ala Phe Ser Ser Asp Leu IleSer Ile His 385 390 395 400 tct cta gca gat gtg gag gtg gtt gtc aca aaactc cat aat gag gat 1248 Ser Leu Ala Asp Val Glu Val Val Val Thr Lys LeuHis Asn Glu Asp 405 410 415 atc aaa gaa gaa gtg tgg ata ggc ctt aag aacata aac ata cca act 1296 Ile Lys Glu Glu Val Trp Ile Gly Leu Lys Asn IleAsn Ile Pro Thr 420 425 430 tta ttt cag tgg tca gat ggt act gaa gtt actcta aca tat tgg gat 1344 Leu Phe Gln Trp Ser Asp Gly Thr Glu Val Thr LeuThr Tyr Trp Asp 435 440 445 gag aat gag cca aat gtt ccc tac aat aag acgccc aac tgt gtt tcc 1392 Glu Asn Glu Pro Asn Val Pro Tyr Asn Lys Thr ProAsn Cys Val Ser 450 455 460 tac tta gga gag cta ggt cag tgg aaa gtc caatca tgt gag gag aaa 1440 Tyr Leu Gly Glu Leu Gly Gln Trp Lys Val Gln SerCys Glu Glu Lys 465 470 475 480 cta aaa tat gta tgc aag aga aag gga gaaaaa ctg aat gac gca agt 1488 Leu Lys Tyr Val Cys Lys Arg Lys Gly Glu LysLeu Asn Asp Ala Ser 485 490 495 tct gat aag atg tgt cct cca gat gag ggctgg aag aga cat gga gaa 1536 Ser Asp Lys Met Cys Pro Pro Asp Glu Gly TrpLys Arg His Gly Glu 500 505 510 acc tgt tac aag att tat gag gat gag gtccct ttt gga aca aac tgc 1584 Thr Cys Tyr Lys Ile Tyr Glu Asp Glu Val ProPhe Gly Thr Asn Cys 515 520 525 aat ctg act atc act agc aga ttt gag caagaa tac cta aat gat ttg 1632 Asn Leu Thr Ile Thr Ser Arg Phe Glu Gln GluTyr Leu Asn Asp Leu 530 535 540 atg aaa aag tat gat aaa tct cta aga aaatac ttc tgg act ggc ctg 1680 Met Lys Lys Tyr Asp Lys Ser Leu Arg Lys TyrPhe Trp Thr Gly Leu 545 550 555 560 aga gat gta gat tct tgt gga gag tataac tgg gca act gtt ggt gga 1728 Arg Asp Val Asp Ser Cys Gly Glu Tyr AsnTrp Ala Thr Val Gly Gly 565 570 575 aga agg cgg gct gta acc ttt tcc aactgg aat ttt ctt gag cca gct 1776 Arg Arg Arg Ala Val Thr Phe Ser Asn TrpAsn Phe Leu Glu Pro Ala 580 585 590 tcc ccg ggc ggc tgc gtg gct atg tctact gga aag tct gtt gga aag 1824 Ser Pro Gly Gly Cys Val Ala Met Ser ThrGly Lys Ser Val Gly Lys 595 600 605 tgg gag gtg aag gac tgc aga agc ttcaaa gca ctt tca att tgc aag 1872 Trp Glu Val Lys Asp Cys Arg Ser Phe LysAla Leu Ser Ile Cys Lys 610 615 620 aaa atg agt gga ccc ctt ggg cct gaagaa gca tcc cct aag cct gat 1920 Lys Met Ser Gly Pro Leu Gly Pro Glu GluAla Ser Pro Lys Pro Asp 625 630 635 640 gac ccc tgt cct gaa ggc tgg cagagt ttc ccc gca agt ctt tct tgt 1968 Asp Pro Cys Pro Glu Gly Trp Gln SerPhe Pro Ala Ser Leu Ser Cys 645 650 655 tat aag gta ttc cat gca gaa agaatt gta aga aag agg aac tgg gaa 2016 Tyr Lys Val Phe His Ala Glu Arg IleVal Arg Lys Arg Asn Trp Glu 660 665 670 gaa gct gaa cga ttc tgc caa gccctt gga gca cac ctt tct agc ttc 2064 Glu Ala Glu Arg Phe Cys Gln Ala LeuGly Ala His Leu Ser Ser Phe 675 680 685 agc cat gtg gat gaa ata aag gaattt ctt cac ttt tta acg gac cag 2112 Ser His Val Asp Glu Ile Lys Glu PheLeu His Phe Leu Thr Asp Gln 690 695 700 ttc agt ggc cag cat tgg ctg tggatt ggt ttg aat aaa agg agc cca 2160 Phe Ser Gly Gln His Trp Leu Trp IleGly Leu Asn Lys Arg Ser Pro 705 710 715 720 gat tta caa gga tcc tgg caatgg agt gat cgt aca cca gtg tct act 2208 Asp Leu Gln Gly Ser Trp Gln TrpSer Asp Arg Thr Pro Val Ser Thr 725 730 735 att atc atg cca aat gag tttcag cag gat tat gac atc aga gac tgt 2256 Ile Ile Met Pro Asn Glu Phe GlnGln Asp Tyr Asp Ile Arg Asp Cys 740 745 750 gct gct gtc aag gta ttt catagg cca tgg cga aga ggc tgg cat ttc 2304 Ala Ala Val Lys Val Phe His ArgPro Trp Arg Arg Gly Trp His Phe 755 760 765 tat gat gat aga gaa ttt atttat ttg agg cct ttt gct tgt gat aca 2352 Tyr Asp Asp Arg Glu Phe Ile TyrLeu Arg Pro Phe Ala Cys Asp Thr 770 775 780 aaa ctt gaa tgg gtg tgc caaatt cca aaa ggc cgt act cca aaa aca 2400 Lys Leu Glu Trp Val Cys Gln IlePro Lys Gly Arg Thr Pro Lys Thr 785 790 795 800 cca gac tgg tac aat ccagac cgt gct gga att cat gga cct cca ctt 2448 Pro Asp Trp Tyr Asn Pro AspArg Ala Gly Ile His Gly Pro Pro Leu 805 810 815 ata att gaa gga agt gaatat tgg ttt gtt gct gat ctt cac cta aac 2496 Ile Ile Glu Gly Ser Glu TyrTrp Phe Val Ala Asp Leu His Leu Asn 820 825 830 tat gaa gaa gcc gtc ctgtac tgt gcc agc aat cac agc ttt ctt gcg 2544 Tyr Glu Glu Ala Val Leu TyrCys Ala Ser Asn His Ser Phe Leu Ala 835 840 845 act ata aca tct ttt gtggga cta aaa gcc atc aaa aac aaa ata gca 2592 Thr Ile Thr Ser Phe Val GlyLeu Lys Ala Ile Lys Asn Lys Ile Ala 850 855 860 aat ata tct ggt gat ggacag aag tgg tgg ata aga att agc gag tgg 2640 Asn Ile Ser Gly Asp Gly GlnLys Trp Trp Ile Arg Ile Ser Glu Trp 865 870 875 880 cca ata gat gat catttt aca tac tca cga tat cca tgg cac cgc ttt 2688 Pro Ile Asp Asp His PheThr Tyr Ser Arg Tyr Pro Trp His Arg Phe 885 890 895 cct gtg aca ttt ggagag gaa tgc ttg tac atg tct gcc aag act tgg 2736 Pro Val Thr Phe Gly GluGlu Cys Leu Tyr Met Ser Ala Lys Thr Trp 900 905 910 ctt atc gac tta ggtaaa cca aca gac tgt agt acc aag ttg ccc ttc 2784 Leu Ile Asp Leu Gly LysPro Thr Asp Cys Ser Thr Lys Leu Pro Phe 915 920 925 atc tgt gaa aaa tataat gtt tct tcg tta gag aaa tac agc cca gat 2832 Ile Cys Glu Lys Tyr AsnVal Ser Ser Leu Glu Lys Tyr Ser Pro Asp 930 935 940 tct gca gct aaa gtgcaa tgt tct gag caa tgg att cct ttt cag aat 2880 Ser Ala Ala Lys Val GlnCys Ser Glu Gln Trp Ile Pro Phe Gln Asn 945 950 955 960 aag tgt ttt ctaaag atc aaa ccc gtg tct ctc aca ttt tct caa gca 2928 Lys Cys Phe Leu LysIle Lys Pro Val Ser Leu Thr Phe Ser Gln Ala 965 970 975 agc gat acc tgtcac tcc tat ggt ggc acc ctt cct tca gtg ttg agc 2976 Ser Asp Thr Cys HisSer Tyr Gly Gly Thr Leu Pro Ser Val Leu Ser 980 985 990 cag att gaa caagac ttt att aca tcc ttg ctt ccg gat atg gaa gct 3024 Gln Ile Glu Gln AspPhe Ile Thr Ser Leu Leu Pro Asp Met Glu Ala 995 1000 1005 act tta tggatt ggt ttg cgc tgg act gcc tat gaa aag ata aac aaa 3072 Thr Leu Trp IleGly Leu Arg Trp Thr Ala Tyr Glu Lys Ile Asn Lys 1010 1015 1020 tgg acagat aac aga gag ctg acg tac agt aac ttt cac cca tta ttg 3120 Trp Thr AspAsn Arg Glu Leu Thr Tyr Ser Asn Phe His Pro Leu Leu 1025 1030 1035 1040gtt agt ggg agg ctg aga ata cca gaa aat ttt ttt gag gaa gag tct 3168 ValSer Gly Arg Leu Arg Ile Pro Glu Asn Phe Phe Glu Glu Glu Ser 1045 10501055 cgc tac cac tgt gcc cta ata ctc aac ctc caa aaa tca ccg ttt act3216 Arg Tyr His Cys Ala Leu Ile Leu Asn Leu Gln Lys Ser Pro Phe Thr1060 1065 1070 ggg acg tgg aat ttt aca tcc tgc agt gaa cgc cac ttt gtgtct ctc 3264 Gly Thr Trp Asn Phe Thr Ser Cys Ser Glu Arg His Phe Val SerLeu 1075 1080 1085 tgt cag aaa tat tca gaa gtt aaa agc aga cag acg ttgcag aat gct 3312 Cys Gln Lys Tyr Ser Glu Val Lys Ser Arg Gln Thr Leu GlnAsn Ala 1090 1095 1100 tca gaa act gta aag tat cta aat aat ctg tac aaaata atc cca aag 3360 Ser Glu Thr Val Lys Tyr Leu Asn Asn Leu Tyr Lys IleIle Pro Lys 1105 1110 1115 1120 act ctg act tgg cac agt gct aaa agg gagtgt ctg aaa agt aac atg 3408 Thr Leu Thr Trp His Ser Ala Lys Arg Glu CysLeu Lys Ser Asn Met 1125 1130 1135 cag ctg gtg agc atc acg gac cct taccag cag gca ttc ctc agt gtg 3456 Gln Leu Val Ser Ile Thr Asp Pro Tyr GlnGln Ala Phe Leu Ser Val 1140 1145 1150 cag gcg ctc ctt cac aac tct tcctta tgg atc gga ctc ttc agt caa 3504 Gln Ala Leu Leu His Asn Ser Ser LeuTrp Ile Gly Leu Phe Ser Gln 1155 1160 1165 gat gat gaa ctc aac ttt ggttgg tca gat ggg aaa cgt ctt cat ttt 3552 Asp Asp Glu Leu Asn Phe Gly TrpSer Asp Gly Lys Arg Leu His Phe 1170 1175 1180 agt cgc tgg gct gaa actaat ggg caa ctc gaa gac tgt gta gta tta 3600 Ser Arg Trp Ala Glu Thr AsnGly Gln Leu Glu Asp Cys Val Val Leu 1185 1190 1195 1200 gac act gat ggattc tgg aaa aca gtt gat tgc aat gac aat caa cca 3648 Asp Thr Asp Gly PheTrp Lys Thr Val Asp Cys Asn Asp Asn Gln Pro 1205 1210 1215 ggt gct atttgc tac tat tca gga aat gag act gaa aaa gag gtc aaa 3696 Gly Ala Ile CysTyr Tyr Ser Gly Asn Glu Thr Glu Lys Glu Val Lys 1220 1225 1230 cca gttgac agt gtt aaa tgt cca tct cct gtt cta aat act ccg tgg 3744 Pro Val AspSer Val Lys Cys Pro Ser Pro Val Leu Asn Thr Pro Trp 1235 1240 1245 atacca ttt cag aac tgt tgc tac aat ttc ata ata aca aag aat agg 3792 Ile ProPhe Gln Asn Cys Cys Tyr Asn Phe Ile Ile Thr Lys Asn Arg 1250 1255 1260cat atg gca aca aca cag gat gaa gtt cat act aaa tgc cag aaa ctg 3840 HisMet Ala Thr Thr Gln Asp Glu Val His Thr Lys Cys Gln Lys Leu 1265 12701275 1280 aat cca aaa tca cat att ctg agt att cga gat gaa aag gag aataac 3888 Asn Pro Lys Ser His Ile Leu Ser Ile Arg Asp Glu Lys Glu Asn Asn1285 1290 1295 ttt gtt ctt gag caa ctg ctg tac ttc aat tat atg gct tcatgg gtc 3936 Phe Val Leu Glu Gln Leu Leu Tyr Phe Asn Tyr Met Ala Ser TrpVal 1300 1305 1310 atg tta gga ata act tat aga aat aat tct ctt atg tggttt gat aag 3984 Met Leu Gly Ile Thr Tyr Arg Asn Asn Ser Leu Met Trp PheAsp Lys 1315 1320 1325 acc cca ctg tca tat aca cat tgg aga gca gga agacca act ata aaa 4032 Thr Pro Leu Ser Tyr Thr His Trp Arg Ala Gly Arg ProThr Ile Lys 1330 1335 1340 aat gag aag ttt ttg gct ggt tta agt act gacggc ttc tgg gat att 4080 Asn Glu Lys Phe Leu Ala Gly Leu Ser Thr Asp GlyPhe Trp Asp Ile 1345 1350 1355 1360 caa acc ttt aaa gtt att gaa gaa gcagtt tat ttt cac cag cac agc 4128 Gln Thr Phe Lys Val Ile Glu Glu Ala ValTyr Phe His Gln His Ser 1365 1370 1375 att ctt gct tgt aaa att gaa atggtt gac tac aaa gaa gaa cat aat 4176 Ile Leu Ala Cys Lys Ile Glu Met ValAsp Tyr Lys Glu Glu His Asn 1380 1385 1390 act aca ctg cca cag ttt atgcca tat gaa gat ggt att tac agt gtt 4224 Thr Thr Leu Pro Gln Phe Met ProTyr Glu Asp Gly Ile Tyr Ser Val 1395 1400 1405 att caa aaa aag gta acatgg tat gaa gca tta aac atg tgt tct caa 4272 Ile Gln Lys Lys Val Thr TrpTyr Glu Ala Leu Asn Met Cys Ser Gln 1410 1415 1420 agt gga ggt cac ttggca agc gtt cac aac caa aat ggc cag ctc ttt 4320 Ser Gly Gly His Leu AlaSer Val His Asn Gln Asn Gly Gln Leu Phe 1425 1430 1435 1440 ctg gaa gatatt gta aaa cgt gat gga ttt cca cta tgg gtt ggg ctc 4368 Leu Glu Asp IleVal Lys Arg Asp Gly Phe Pro Leu Trp Val Gly Leu 1445 1450 1455 tca agtcat gat gga agt gaa tca agt ttt gaa tgg tct gat ggt agt 4416 Ser Ser HisAsp Gly Ser Glu Ser Ser Phe Glu Trp Ser Asp Gly Ser 1460 1465 1470 acattt gac tat atc cca tgg aaa ggc caa aca tct cct gga aat tgt 4464 Thr PheAsp Tyr Ile Pro Trp Lys Gly Gln Thr Ser Pro Gly Asn Cys 1475 1480 1485gtt ctc ttg gat cca aaa gga act tgg aaa cat gaa aaa tgc aac tct 4512 ValLeu Leu Asp Pro Lys Gly Thr Trp Lys His Glu Lys Cys Asn Ser 1490 14951500 gtt aag gat ggt gct att tgt tat aaa cct aca aaa tct aaa aag ctg4560 Val Lys Asp Gly Ala Ile Cys Tyr Lys Pro Thr Lys Ser Lys Lys Leu1505 1510 1515 1520 tcc cgt ctt aca tat tca tca aga tgt cca gca gca aaagag aat ggg 4608 Ser Arg Leu Thr Tyr Ser Ser Arg Cys Pro Ala Ala Lys GluAsn Gly 1525 1530 1535 tca cgg tgg atc cag tac aag ggt cac tgt tac aagtct gat cag gca 4656 Ser Arg Trp Ile Gln Tyr Lys Gly His Cys Tyr Lys SerAsp Gln Ala 1540 1545 1550 ttg cac agt ttt tca gag gcc aaa aaa ttg tgttca aaa cat gat cac 4704 Leu His Ser Phe Ser Glu Ala Lys Lys Leu Cys SerLys His Asp His 1555 1560 1565 tct gca act atc gtt tcc ata aaa gat gaagat gag aat aaa ttt gtg 4752 Ser Ala Thr Ile Val Ser Ile Lys Asp Glu AspGlu Asn Lys Phe Val 1570 1575 1580 agc aga ctg atg agg gaa aat aat aacatt acc atg aga gtt tgg ctt 4800 Ser Arg Leu Met Arg Glu Asn Asn Asn IleThr Met Arg Val Trp Leu 1585 1590 1595 1600 gga tta tct caa cat tct gttgac cag tct tgg agt tgg tta gat gga 4848 Gly Leu Ser Gln His Ser Val AspGln Ser Trp Ser Trp Leu Asp Gly 1605 1610 1615 tca gaa gtg aca ttt gtcaaa tgg gaa aat aaa agt aag agt ggt gtt 4896 Ser Glu Val Thr Phe Val LysTrp Glu Asn Lys Ser Lys Ser Gly Val 1620 1625 1630 gga aga tgt agc atgttg ata gct tca aat gaa act tgg aaa aaa gtt 4944 Gly Arg Cys Ser Met LeuIle Ala Ser Asn Glu Thr Trp Lys Lys Val 1635 1640 1645 gaa tgt gaa catggt ttt gga aga gtt gtc tgc aaa gtg cct ctg ggc 4992 Glu Cys Glu His GlyPhe Gly Arg Val Val Cys Lys Val Pro Leu Gly 1650 1655 1660 cct gat tacaca gca ata gct atc ata gtt gcc aca cta agt atc tta 5040 Pro Asp Tyr ThrAla Ile Ala Ile Ile Val Ala Thr Leu Ser Ile Leu 1665 1670 1675 1680 gttctc atg ggc gga ctg att tgg ttc ctc ttc caa agg cac cgt ttg 5088 Val LeuMet Gly Gly Leu Ile Trp Phe Leu Phe Gln Arg His Arg Leu 1685 1690 1695cac ctg gcg ggt ttc tca tca gtt cga tat gca caa gga gtg aat gaa 5136 HisLeu Ala Gly Phe Ser Ser Val Arg Tyr Ala Gln Gly Val Asn Glu 1700 17051710 gat gag att atg ctt cct tct ttc cat gac taa 5169 Asp Glu Ile MetLeu Pro Ser Phe His Asp 1715 1720 3 349 DNA Homo sapiens 3 aacagttgattgcaatgaca atcaaccagg tgctatttgc tactattcag gaaatgagac 60 tgaaaaagaggtcaaaccag ttgacagtgt taaatgtcca tctcctgttc taaatactcc 120 gtggataccatttcagaact gttgctacaa tttcataata acaaagaata ggcatatggc 180 aacaacacaggatgaagttc atactaaatg ccagaaactg aatccaaaat cacatattct 240 gagtattcgagatgaaaagg agaataactt tgttcttgag caactgctgt acttcaatta 300 tatggcttcatgggtcatgt taggaataac ttatagaaat aaktctctt 349 4 152 DNA Homo sapiens 4attaatatgc tgtggaagtg ggtgtcccag catcggctct ttcatttgca ctcccaaaag 60tgccttggcc tcgatattac caaatcggta aatgagctga gaatgttcag ctgtgactcc 120agtgccatgc tgtggtggaa atgcgagcac ca 152 5 20 DNA Artificial SequenceDescription of Artificial Sequence Primer 5 gaycangayg gnttytggaa 20 620 DNA Artificial Sequence Description of Artificial Sequence Primer 6tacaccaarc trttytgncg 20 7 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 7 aayatgctnt ggaartgggt 20 8 20 DNAArtificial Sequence Description of Artificial Sequence Primer 8tgrtgytcrc ayttccacca 20 9 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 9 gayacngayg gnttytggaa 20 10 20 DNAArtificial Sequence Description of Artificial Sequence Primer 10gcngtyttrt craaccacat 20 11 26 DNA Artificial Sequence Description ofArtificial Sequence Primer 11 gctctagaaa catgacccat gaagcc 26 12 27 DNAArtificial Sequence Description of Artificial Sequence Primer 12gctctagaca tcggctcttt catttgt 27 13 27 DNA Artificial SequenceDescription of Artificial Sequence Primer 13 cgggattcac agttgattgcaatgaca 27 14 35 DNA Artificial Sequence Description of ArtificialSequence Oligo d(T) adaptor primer 14 gactagtctg cagaattctt ttttttttttttttt 35 15 18 DNA Artificial Sequence Description of ArtificialSequence Adaptor primer 15 gactagtctg cagaattc 18 16 28 DNA ArtificialSequence Description of Artificial Sequence Primer 16 cgggatccctctggccgcgc actaatga 28 17 31 DNA Artificial Sequence Description ofArtificial Sequence Primer 17 ccgctcgagc tgtggatacc agcacatgcc t 31 1824 DNA Artificial Sequence Description of Artificial Sequence Primer 18gatgggaact cttatgggag acct 24 19 24 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 19 tgatgcaggc tggctgccaa ataa 24 20 24 DNAArtificial Sequence Description of Artificial Sequence Primer 20aactgggcaa ctgttggtgg aaga 24 21 24 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 21 atggcgaaga ggctggcatt tcta 24 22 24 DNAArtificial Sequence Description of Artificial Sequence Primer 22ctcaagcaag cgatacctgt cact 24 23 24 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 23 tgggcaactc gaagactgtg tagt 24 24 24 DNAArtificial Sequence Description of Artificial Sequence Primer 24caccagcaca gcattcttgc ttgt 24 25 24 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 25 atttgtgagc agactgatga ggga 24 26 32 DNAArtificial Sequence Description of Artificial Sequence PCR-fragment 26cggaattcga tctcatgata aggctggtca ca 32 27 21 DNA Artificial SequenceDescription of Artificial Sequence Primer 060 27 gtggatccag tacaagggtc a21 28 21 DNA Artificial Sequence Description of Artificial SequencePrimer 056 28 accaaatcag tccgcccatg a 21 29 21 DNA Artificial SequenceDescription of Artificial Sequence Primer 053 29 atggggaagg tgaaggtcgg a21 30 21 DNA Artificial Sequence Description of Artificial SequencePrimer 053 30 aggggccatc cacagtcttc t 21 31 1723 PRT Murine sp. 31 MetArg Thr Gly Arg Val Thr Pro Gly Leu Ala Ala Gly Leu Leu Leu 1 5 10 15Leu Leu Leu Arg Ser Phe Gly Leu Val Glu Pro Ser Glu Ser Ser Gly 20 25 30Asn Asp Pro Phe Thr Ile Val His Glu Asn Thr Gly Lys Cys Ile Gln 35 40 45Pro Leu Ser Asp Trp Val Val Ala Gln Asp Cys Ser Gly Thr Asn Asn 50 55 60Met Leu Trp Lys Trp Val Ser Gln His Arg Leu Phe His Leu Glu Ser 65 70 7580 Gln Lys Cys Leu Gly Leu Asp Ile Thr Lys Ala Thr Asp Asn Leu Arg 85 9095 Met Phe Ser Cys Asp Ser Thr Val Met Leu Trp Trp Lys Cys Glu His 100105 110 His Ser Leu Tyr Thr Ala Ala Gln Tyr Arg Leu Ala Leu Lys Asp Gly115 120 125 Tyr Ala Val Ala Asn Thr Asn Thr Ser Asp Val Trp Lys Lys GlyGly 130 135 140 Ser Glu Glu Asn Leu Cys Ala Gln Pro Tyr His Glu Ile TyrThr Arg 145 150 155 160 Asp Gly Asn Ser Tyr Gly Arg Pro Cys Glu Phe ProPhe Leu Ile Gly 165 170 175 Glu Thr Trp Tyr His Asp Cys Ile His Asp GluAsp His Ser Gly Pro 180 185 190 Trp Cys Ala Thr Thr Leu Ser Tyr Glu TyrAsp Gln Lys Trp Gly Ile 195 200 205 Cys Leu Leu Pro Glu Ser Gly Cys GluGly Asn Trp Glu Lys Asn Glu 210 215 220 Gln Ile Gly Ser Cys Tyr Gln PheAsn Asn Gln Glu Ile Leu Ser Trp 225 230 235 240 Lys Glu Ala Tyr Val SerCys Gln Asn Gln Gly Ala Asp Leu Leu Ser 245 250 255 Ile His Ser Ala AlaGlu Leu Ala Tyr Ile Thr Gly Lys Glu Asp Ile 260 265 270 Ala Arg Leu ValTrp Leu Gly Leu Asn Gln Leu Tyr Ser Ala Arg Gly 275 280 285 Trp Glu TrpSer Asp Phe Arg Pro Leu Lys Phe Leu Asn Trp Asp Pro 290 295 300 Gly ThrPro Val Ala Pro Val Ile Gly Gly Ser Ser Cys Ala Arg Met 305 310 315 320Asp Thr Glu Ser Gly Leu Trp Gln Ser Val Ser Cys Glu Ser Gln Gln 325 330335 Pro Tyr Val Cys Lys Lys Pro Leu Asn Asn Thr Leu Glu Leu Pro Asp 340345 350 Val Trp Thr Tyr Thr Asp Thr His Cys His Val Gly Trp Leu Pro Asn355 360 365 Asn Gly Phe Cys Tyr Leu Leu Ala Asn Glu Ser Ser Ser Trp AspAla 370 375 380 Ala His Leu Lys Cys Lys Ala Phe Gly Ala Asp Leu Ile SerMet His 385 390 395 400 Ser Leu Ala Asp Val Glu Val Val Val Thr Lys LeuHis Asn Gly Asp 405 410 415 Val Lys Lys Glu Ile Trp Thr Gly Leu Lys AsnThr Asn Ser Pro Ala 420 425 430 Leu Phe Gln Trp Ser Asp Gly Thr Glu ValThr Leu Thr Tyr Trp Asn 435 440 445 Glu Asn Glu Pro Ser Val Pro Phe AsnLys Thr Pro Asn Cys Val Ser 450 455 460 Tyr Leu Gly Lys Leu Gly Gln TrpLys Val Gln Ser Cys Glu Lys Lys 465 470 475 480 Leu Arg Tyr Val Cys LysLys Lys Gly Glu Ile Thr Lys Asp Ala Glu 485 490 495 Ser Asp Lys Leu CysPro Pro Asp Glu Gly Trp Lys Arg His Gly Glu 500 505 510 Thr Cys Tyr LysIle Tyr Glu Lys Glu Ala Pro Phe Gly Thr Asn Cys 515 520 525 Asn Leu ThrIle Thr Ser Arg Phe Glu Gln Glu Phe Leu Asn Tyr Met 530 535 540 Met LysAsn Tyr Asp Lys Ser Leu Arg Lys Tyr Phe Trp Thr Gly Leu 545 550 555 560Arg Asp Pro Asp Ser Arg Gly Glu Tyr Ser Trp Ala Val Ala Gln Gly 565 570575 Val Lys Gln Ala Val Thr Phe Ser Asn Trp Asn Phe Leu Glu Pro Ala 580585 590 Ser Pro Gly Gly Cys Val Ala Met Ser Thr Gly Lys Thr Leu Gly Lys595 600 605 Trp Glu Val Lys Asn Cys Arg Ser Phe Arg Ala Leu Ser Ile CysLys 610 615 620 Lys Val Ser Glu Pro Gln Glu Pro Glu Glu Ala Ala Pro LysPro Asp 625 630 635 640 Asp Pro Cys Pro Glu Gly Trp His Thr Phe Pro SerSer Leu Ser Cys 645 650 655 Tyr Lys Val Phe His Ile Glu Arg Ile Val ArgLys Arg Asn Trp Glu 660 665 670 Glu Ala Glu Arg Phe Cys Gln Ala Leu GlyAla His Leu Pro Ser Phe 675 680 685 Ser Arg Arg Glu Glu Ile Lys Asp PheVal His Leu Leu Lys Asp Gln 690 695 700 Phe Ser Gly Gln Arg Trp Leu TrpIle Gly Leu Asn Lys Arg Ser Pro 705 710 715 720 Asp Leu Gln Gly Ser TrpGln Trp Ser Asp Arg Thr Pro Val Ser Ala 725 730 735 Val Met Met Glu ProGlu Phe Gln Gln Asp Phe Asp Ile Arg Asp Cys 740 745 750 Ala Ala Ile LysVal Leu Asp Val Pro Trp Arg Arg Val Trp His Leu 755 760 765 Tyr Glu AspLys Asp Tyr Ala Tyr Trp Lys Pro Phe Ala Cys Asp Ala 770 775 780 Lys LeuGlu Trp Val Cys Gln Ile Pro Lys Gly Ser Thr Pro Gln Met 785 790 795 800Pro Asp Trp Tyr Asn Pro Glu Arg Thr Gly Ile His Gly Pro Pro Val 805 810815 Ile Ile Glu Gly Ser Glu Tyr Trp Phe Val Ala Asp Pro His Leu Asn 820825 830 Tyr Glu Glu Ala Val Leu Tyr Cys Ala Ser Asn His Ser Phe Leu Ala835 840 845 Thr Ile Thr Ser Phe Thr Gly Leu Lys Ala Ile Lys Asn Lys LeuAla 850 855 860 Asn Ile Ser Gly Glu Glu Gln Lys Trp Trp Val Lys Thr SerGlu Asn 865 870 875 880 Pro Ile Asp Arg Tyr Phe Leu Gly Ser Arg Arg ArgLeu Trp His His 885 890 895 Phe Pro Met Thr Phe Gly Asp Glu Cys Leu HisMet Ser Ala Lys Thr 900 905 910 Trp Leu Val Asp Leu Ser Lys Arg Ala AspCys Asn Ala Lys Leu Pro 915 920 925 Phe Ile Cys Glu Arg Tyr Asn Val SerSer Leu Glu Lys Tyr Ser Pro 930 935 940 Asp Pro Ala Ala Lys Val Gln CysThr Glu Lys Trp Ile Pro Phe Gln 945 950 955 960 Asn Lys Cys Phe Leu LysVal Asn Ser Gly Pro Val Thr Phe Ser Gln 965 970 975 Ala Ser Gly Ile CysHis Ser Tyr Gly Gly Thr Leu Pro Ser Val Leu 980 985 990 Ser Arg Gly GluGln Asp Phe Ile Ile Ser Leu Leu Pro Glu Met Glu 995 1000 1005 Ala SerLeu Trp Ile Gly Leu Arg Trp Thr Ala Tyr Glu Arg Ile Asn 1010 1015 1020Arg Trp Thr Asp Asn Arg Glu Leu Thr Tyr Ser Asn Phe His Pro Leu 10251030 1035 1040 Leu Val Gly Arg Arg Leu Ser Ile Pro Thr Asn Phe Phe AspAsp Glu 1045 1050 1055 Ser His Phe His Cys Ala Leu Ile Leu Asn Leu LysLys Ser Pro Leu 1060 1065 1070 Thr Gly Thr Trp Asn Phe Thr Ser Cys SerGlu Arg His Ser Leu Ser 1075 1080 1085 Leu Cys Gln Lys Tyr Ser Glu ThrGlu Asp Gly Gln Pro Trp Glu Asn 1090 1095 1100 Thr Ser Lys Thr Val LysTyr Leu Asn Asn Leu Tyr Lys Ile Ile Ser 1105 1110 1115 1120 Lys Pro LeuThr Trp His Gly Ala Leu Lys Glu Cys Met Lys Glu Lys 1125 1130 1135 MetArg Leu Val Ser Ile Thr Asp Pro Tyr Gln Gln Ala Phe Leu Ala 1140 11451150 Val Gln Ala Thr Leu Arg Asn Ser Ser Phe Trp Ile Gly Leu Ser Ser1155 1160 1165 Gln Asp Asp Glu Leu Asn Phe Gly Trp Ser Asp Gly Lys ArgLeu Gln 1170 1175 1180 Phe Ser Asn Trp Ala Gly Ser Asn Glu Gln Leu AspAsp Cys Val Ile 1185 1190 1195 1200 Leu Asp Thr Asp Gly Phe Trp Lys ThrAla Asp Cys Asp Asp Asn Gln 1205 1210 1215 Pro Gly Ala Ile Cys Tyr TyrPro Gly Asn Glu Thr Glu Glu Glu Val 1220 1225 1230 Arg Ala Leu Asp ThrAla Lys Cys Pro Ser Pro Val Gln Ser Thr Pro 1235 1240 1245 Trp Ile ProPhe Gln Asn Ser Cys Tyr Phe Asn Met Ile Thr Asn Asn 1250 1255 1260 ArgHis Lys Thr Val Thr Pro Glu Glu Val Gln Ser Thr Cys Glu Lys 1265 12701275 1280 Leu His Pro Lys Ala His Ser Leu Ser Ile Arg Asn Glu Glu GluAsn 1285 1290 1295 Thr Phe Val Val Glu Gln Leu Leu Tyr Phe Asn Tyr IleAla Ser Trp 1300 1305 1310 Val Met Leu Gly Ile Thr Tyr Glu Asn Asn SerLeu Met Trp Phe Asp 1315 1320 1325 Lys Thr Ala Leu Ser Tyr Thr His TrpArg Thr Gly Arg Pro Thr Val 1330 1335 1340 Lys Asn Gly Lys Phe Leu AlaGly Leu Ser Thr Asp Gly Phe Trp Asp 1345 1350 1355 1360 Ile Gln Ser PheAsn Val Ile Glu Glu Thr Leu His Phe Tyr Gln His 1365 1370 1375 Ser IleSer Ala Cys Lys Ile Lys Met Val Asp Tyr Glu Asp Lys His 1380 1385 1390Asn Gly Thr Leu Pro Gln Phe Ile Pro Tyr Lys Asp Gly Val Tyr Ser 13951400 1405 Val Ile Gln Lys Lys Val Thr Trp Tyr Glu Ala Leu Asn Ala CysSer 1410 1415 1420 Gln Ser Gly Gly Glu Leu Ala Ser Val His Asn Pro AsnGly Lys Leu 1425 1430 1435 1440 Phe Leu Glu Asp Ile Val Asn Arg Asp GlyPhe Pro Leu Asn Val Gly 1445 1450 1455 Leu Ser Ser His Asp Gly Ser GluSer Ser Phe Glu Trp Ser Asp Gly 1460 1465 1470 Arg Ala Phe Asp Tyr ValPro Trp Gln Ser Leu Gln Ser Pro Gly Asp 1475 1480 1485 Cys Val Val LeuTyr Pro Lys Gly Ile Trp Arg Arg Glu Lys Cys Leu 1490 1495 1500 Ser ValLys Asp Gly Ala Ile Cys Tyr Lys Pro Thr Lys Asp Lys Lys 1505 1510 15151520 Leu Ile Phe His Val Lys Ser Ser Lys Cys Pro Val Ala Lys Arg Asp1525 1530 1535 Gly Pro Gln Trp Val Gln Tyr Gly Gly His Cys Tyr Ala SerAsp Gln 1540 1545 1550 Val Leu His Ser Phe Ser Glu Ala Lys Gln Val CysGln Glu Leu Asp 1555 1560 1565 His Ser Ala Thr Val Val Thr Ile Ala AspGlu Asn Glu Asn Lys Phe 1570 1575 1580 Val Ser Arg Leu Met Arg Glu AsnTyr Asn Ile Thr Met Arg Val Trp 1585 1590 1595 1600 Leu Gly Leu Ser GlnHis Ser Leu Asp Gln Ser Trp Ser Trp Leu Asp 1605 1610 1615 Gly Leu AspVal Thr Phe Val Lys Trp Glu Asn Lys Thr Lys Asp Gly 1620 1625 1630 AspGly Lys Cys Ser Ile Leu Ile Ala Ser Asn Glu Thr Trp Arg Lys 1635 16401645 Val His Cys Ser Arg Gly Tyr Ala Arg Ala Val Cys Lys Ile Pro Leu1650 1655 1660 Ser Pro Asp Tyr Thr Gly Ile Ala Ile Leu Phe Ala Val LeuCys Leu 1665 1670 1675 1680 Leu Gly Leu Ile Ser Leu Ala Ile Trp Phe LeuLeu Gln Arg Ser His 1685 1690 1695 Ile Arg Trp Thr Gly Phe Ser Ser ValArg Tyr Glu His Gly Thr Asn 1700 1705 1710 Glu Asp Glu Val Met Leu ProSer Phe His Asp 1715 1720

1. Isolated human DEC-205 which has an approximate molecular weight of198-205 kDa and which includes the following amino acid sequences: (i)TVDCNDNQPGAICYYSGNETEKEVKPVDSVKCPSPVLNTPWIPFQNCCYNFIITKNRHMATTQDEVQSTCEKLHPKSHILSIRDEKENNFVLEQLLYFNYMASWVMLGITYRNNSL; and (ii)SQHRLFHLHSQKCLGLDITKSVNELRMFSCDSSAML; or a functionally equivalentfragment thereof.
 2. Isolated human DEC-205 which comprises the aminoacid sequence of FIG. 11, or a functionally equivalent fragment thereof.3. Isolated human DEC-205 in mature form which comprises amino acids 27to 1722 of the amino acid sequence of FIG.
 11. 4. The extracellulardomain of human DEC-205 or a functionally equivalent fragment thereof asclaimed in claim
 1. 5. The extracellular domain of human DEC-205 havingan amino acid sequence which includes amino acids 27 to 1661 of FIG. 11or a functionally equivalent fragment thereof.
 6. An extracellulardomain fragment as claimed in claim 5 which includes amino acids 1208 to1323 of amino acid sequence of FIG.
 11. 7. A polynucleotide encodinghuman DEC-205, its extracellular domain or a fragment thereof as definedin any one of claims 1 to
 6. 8. A polynucleotide as claimed in claim 7which includes the following nucleotide sequences: (iii)   A ACA GTT GATTGC AAT GAC AAT CAA CCA GGT GCT ATT TGC TAC TAT TCA GGA AAT GAG ACT GAAAAA GAG GTC AAA CCA GTT GAC AGT GTT AAA TGT CCA TCT CCT GTT CTA AAT ACTCCG TGG ATA CCA TTT CAG AAC TGT TGC TAC AAT TTC ATA ATA ACA AAG AAT AGGCAT ATG GCA ACA ACA CAG GAT GAA GTT CAT ACT AAA TGC CAG AAA CTG AAT CCAAAA TCA CAT ATT CTG AGT ATT CGA GAT GAA AAG GAG AAT AAC TTT GTT CTT GAGCAA CTG CTG TAC TTC AAT TAT ATG GCT TCA TGG GTC ATG TTA GGA ATA ACT TATAGA AAT AAX TCT CTT; and (iv) ATT AAT ATG CTG TGG AAG TGG GTG TCC CAGCAT CGG CTC TTT CAT TTG CAC TCC CAA AAG TGC CTT GGC CTC GAT ATT ACC AAATCG GTA AAT GAG CTG AGA ATG TTC AGC TGT GAC TCC AGT GCC ATG CTG TGG TGGAAA TGC GAG CAC CA

wherein X is T or G.
 9. A polynucleotide as claimed in claim 7 whichcomprises all of the nucleotide sequence of FIG.
 10. 10. Apolynucleotide as claimed in claim 7 which comprises nucleotides 64 to5166 of the nucleotide sequence of FIG.
 10. 11. A polynucleotide asclaimed in any one of claims 7 to 10 which is DNA.
 12. A vector whichincludes a polynucleotide as claimed in claim
 12. 13. A method ofproducing human DEC-205, an extracellular domain thereof or afunctionally equivalent fragment comprising the steps of: (a) culturinga host cell which has been transformed or transfected with a vector asdefined above to express the encoded human DEC-205, extracellular domainor fragment; and (b) recovering the expressed human DEC-205,extracellular domain or fragment
 14. A ligand that binds to humanDEC-205 or a fragment thereof as claimed in any one of claims 1 to 3.15. A ligand that binds to an extracellular domain of human DEC-205 orfragment thereof as claimed in any one of claims 4 to
 6. 16. A ligand asclaimed in claim 14 or claim 15 which is an antibody, or antibodybinding fragment.
 17. A construct for use in prophylaxis or therapycomprising a ligand as claimed in any one of claims 14 to 16 coupled toan antigen capable of inducing protective immune response in a patient.18. A construct for use in prophylaxis or therapy comprising a ligand asclaimed in any one of claims 14 to 16 coupled to a toxin.
 19. Aconstruct for use in prophylaxis or therapy comprising human DEC-205 oran extracellular domain thereof as claimed in any one of claims 1 to 6coupled to an antigen capable of inducing a protective immune responsein a patient.
 20. A construct for use in prophylaxis or therapycomprising human DEC-205 or an extracellular domain thereof as claimedin any one of claims 1 to 6 coupled to a toxin.
 21. A method ofprophylaxis or therapy which comprises administering to a patient inneed of the same human DEC-205 as claimed in any one of claims 1 to 3,an extracellular domain as claimed in any one of claims 4 to 6, a ligandas claimed in any one of claims 14 to 16, or a construct as claimed inany one of claims 17 to
 20. 22. A process for isolating activateddendritic cells expressing human DEC-205 on the surface thereofcomprising the step of contacting a sample containing said cells with aligand as claimed in claim 16, and isolating those cells to which theligand has bound.